Biological methods for preparing a fatty dicarboxylic acid

ABSTRACT

The technology relates in part to biological methods for producing a fatty dicarboxylic acid and engineered microorganisms capable of such production.

RELATED PATENT APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 61/505,092 filed on Jul. 6, 2011, entitled “BIOLOGICALMETHODS FOR PREPARING SEBACIC ACID” naming Stephen Picataggio and TomBeardslee as inventors, and designated by Attorney Docket No.VRD-1005-PV. This patent application also claims the benefit of U.S.provisional patent application No. 61/523,216 filed Aug. 12, 2011,entitled “BIOLOGICAL METHODS FOR PREPARING DODECANEDIOIC ACID” namingStephen Picataggio and Tom Beardslee as inventors, and designated byAttorney Docket No. VRD-1006-PV. The entire content of each of theforegoing provisional patent applications is incorporated herein byreference, including all text, tables and drawings.

FIELD

The technology relates in part to biological methods for producing afatty dicarboxylic acid and engineered microorganisms capable of suchproduction.

BACKGROUND

Microorganisms employ various enzyme-driven biological pathways tosupport their own metabolism and growth. A cell synthesizes nativeproteins, including enzymes, in vivo from deoxyribonucleic acid (DNA).DNA first is transcribed into a complementary ribonucleic acid (RNA)that comprises a ribonucleotide sequence encoding the protein. RNA thendirects translation of the encoded protein by interaction with variouscellular components, such as ribosomes. The resulting enzymesparticipate as biological catalysts in pathways involved in productionof molecules by the organism.

These pathways can be exploited for the harvesting of the naturallyproduced products. The pathways also can be altered to increaseproduction or to produce different products that may be commerciallyvaluable. Advances in recombinant molecular biology methodology allowresearchers to isolate DNA from one organism and insert it into anotherorganism, thus altering the cellular synthesis of enzymes or otherproteins. Advances in recombinant molecular biology methodology alsoallow endogenous genes, carried in the genomic DNA of a microorganism,to be increased in copy number, thus altering the cellular synthesis ofenzymes or other proteins. Such genetic engineering can change thebiological pathways within the host organism, causing it to produce adesired product. Microorganic industrial production can minimize the useof caustic chemicals and the production of toxic byproducts, thusproviding a “clean” source for certain compounds. The use of appropriateplant derived feedstocks allows production of “green” compounds whilefurther minimizing the need for and use of petroleum derived compounds.

SUMMARY

Provided in certain aspects is a genetically modified yeast, comprising:one or more genetic modifications that substantially block betaoxidation activity; and one or more genetic modifications that increaseone or more activities chosen from monooxygenase activity, monooxygenasereductase activity, thioesterase activity, acyltransferase activity,isocitrate dehydrogenase activity, glyceraldehyde-3-phosphatedehydrogenase activity, glucose-6-phosphate dehydrogenase activity,acyl-coA oxidase activity, fatty alcohol oxidase activity, acyl-CoAhydrolase activity, alcohol dehydrogenase activity, peroxisomalbiogenesis factor activity, and fatty aldehyde dehydrogenase activity.

The one or more genetic modifications sometimes increase one or more of:(a) one or more monooxygase activities chosen from monooxygenaseactivity chosen from CYP52A12 monooxygenase activity, CYP52A13monooxygenase activity, CYP52A14 monooxygenase activity, CYP52A15monooxygenase activity, CYP52A16 monooxygenase activity, CYP52A17monooxygenase activity, CYP52A18 monooxygenase activity, CYP52A19monooxygenase activity, CYP52A20 monooxygenase activity, CYP52D2monooxygenase activity and BM3 monooxygenase activity; (b) one or moremonooxygenase reductase activities chosen from CPRA monooxygenasereductase activity, CPRB monooxygenase reductase activity and CPR750monooxygenase reductase activity; (c) an IDP2 isocitrate dehydrogenaseactivity; (d) a GDP1 glyceraldehyde-3-phosphate dehydrogenase activity;(e) one or more glucose-6-phosphate dehydrogenase activities chosen froma ZWF1 glucose-6-phosphate dehydrogenase activity and ZWF2glucose-6-phosphate dehydrogenase activity; (f) one or more fattyalcohol oxidase activities chosen from FAO1 fatty alcohol oxidaseactivity, FAO2A fatty alcohol oxidase activity, FAO2B fatty alcoholoxidase activity, FAO13 fatty alcohol oxidase activity, FAO17 fattyalcohol oxidase activity, FAO18 fatty alcohol oxidase activity and FAO20fatty alcohol oxidase activity; (g) one or more alcohol dehydrogenaseactivities chosen from ADH1 alcohol dehydrogenase activity, ADH2 alcoholdehydrogenase activity, ADH3 alcohol dehydrogenase activity, ADH4alcohol dehydrogenase activity, ADH5 alcohol dehydrogenase activity,ADH7 alcohol dehydrogenase activity, ADH8 alcohol dehydrogenase activityand SFA alcohol dehydrogenase activity; (h) one or more acyl-CoAhydrolase activities chosen from ACH-A acyl-CoA hydrolase activity andACH-B acyl-CoA hydrolase activity; (i) one or more acyltransferaseactivities chosen from acyl-CoA sterol acyltransferase activity,diacylglycerol acyltransferase activity and phospholipid:diacylglycerolacyltransferase activity; (j) one or more acyltransferase activitieschosen from ARE1 acyl-CoA sterol acyltransferase activity, ARE2 acyl-CoAsterol acyltransferase activity, DGA1 diacylglycerol acyltransferaseactivity, and LRO1 phospholipid:diacylglycerol acyltransferase activity;(k) an acyl-coA thioesterase activity (e.g., a TESA acyl-coAthioesterase activity); (I) a PEX11 peroxisomal biogenesis factoractivity; (m) one or more fatty aldehyde dehydrogenase activities chosenfrom HFD1 fatty aldehyde dehydrogenase activity and HFD2 fatty aldehydedehydrogenase activity; and (n) a POX5 acyl-coA oxidase activity.

In certain aspects, a genetically modified yeast is fully beta oxidationblocked. In some cases all alleles of polynucleotides encoding apolypeptide having acyl-coA oxidase activity are disrupted in agenetically modified yeast. In certain cases where a geneticallymodified yeast is a Candida spp. yeast, all alleles of POX4 and POX5 aredisrupted.

In some aspects, a genetic modification that increases an activity in agenetically modified yeast comprises incorporating in the yeast multiplecopies of a polynucleotide that encodes a polypeptide having theactivity. Sometimes a genetic modification that increases an activity ina genetically modified yeast comprises incorporating in the yeast apromoter in operable linkage with a polynucleotide that encodes apolypeptide having the activity. In some cases the promoter is chosenfrom a POX4 promoter, PEX11 promoter, TEF1 promoter, PGK promoter andFAO1 promoter.

In certain aspects, a genetically modified yeast comprises one or moregenetic modifications that decrease an acyl-coA synthetase activity. Insome cases the one or more genetic modifications decrease one or moreacyl-coA synthetase activities chosen from an ACS1 acyl-coA synthetaseactivity and a FAT1 long-chain acyl-CoA synthetase activity.

In some aspects, a genetically modified yeast is chosen from a Candidaspp. yeast (e.g., C. tropicalis, C. viswanathii, genetically modifiedATCC20336 yeast), Yarrowia spp. yeast, Pichia spp. yeast, Saccharomycesspp. yeast and Kluyveromyces spp. yeast.

Any suitable combination of genetic modifications described herein canbe incorporated into a genetically modified yeast for production of adiacid target product. In some cases, a genetically modified yeastincludes one or more of (a) a genetic modification that increases anactivity, (b) a genetic modification that decreases an activity, and (c)a promoter insertion, as described herein, in any suitable combination.

In some aspects, provided is a method for producing a diacid,comprising: contacting a genetically modified yeast described hereinwith a feedstock capable of being converted by the yeast to a diacid;and culturing the yeast under conditions in which the diacid is producedfrom the feedstock. In some cases the feedstock comprises one or morecomponents from a vegetable oil, and sometimes the diacid is a C4 to C24diacid.

In certain aspects, provided is a method for producing a diacid by ayeast from a feedstock toxic to the yeast, comprising: (a) contacting agenetically modified yeast in culture with a feedstock not substantiallytoxic to the yeast, thereby performing an induction; and (b) contactingthe yeast after the induction in (a) with a feedstock toxic to theyeast, whereby a diacid is produced by the yeast from the feedstocktoxic to the yeast in an amount greater than the amount of the diacidproduced from the feedstock toxic to the yeast when the induction is notperformed.

Provided also herein in some aspects are particular isolated nucleicacids.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 is a schematic representation of the conversion of decane tosebacic acid in a beta-oxidation blocked microorganism. Capric acid isformed as an intermediate during omega oxidation.

FIG. 2 is a schematic representation of the conversion of dodecane tododecanedioic acid in a beta-oxidation blocked microorganism. Lauricacid is formed as an intermediate during omega oxidation.

FIG. 3 is a schematic representation of the conversion of a feedstockcontaining mixed chain-length alkanes to mixed diacids products,including sebacic acid in a beta-oxidation blocked microorganism. Mixedchain-length fatty acids are formed as intermediates during omegaoxidation. Sebacic acid can be separated from other diacid products bythe use of appropriate separation techniques.

FIG. 4 is a schematic representation of the conversion of a feedstockcontaining mixed chain-length alkanes to mixed diacids products,including dodecanedioic acid in a beta-oxidation blocked microorganism.Mixed chain-length fatty acids are formed as intermediates during omegaoxidation. Dodecanedioic acid can be separated from other diacidproducts by the use of appropriate separation techniques.

FIG. 5 is a schematic representation of the conversion of a long-chainalkane into sebacic acid in a partially beta-oxidation blockedmicroorganism. The long-chain alkane is first converted into along-chain fatty acid and then into a long-chain diacid by activities inthe omega-oxidation pathway. The long-chain diacid can be converted tosebacic acid by activities in the beta-oxidation pathway, with thesimultaneous generation of acetyl-CoA.

FIG. 6 is a schematic representation of the conversion of a long-chainalkane into dodecanedioic acid in a partially beta-oxidation blockedmicroorganism. The long-chain alkane is first converted into along-chain fatty acid and then into a long-chain diacid by activities inthe omega-oxidation pathway. The long-chain diacid can be converted tododecanedioic acid by activities in the beta-oxidation pathway, with thesimultaneous generation of acetyl-CoA.

FIG. 7 is a schematic representation of the conversion of a feedstockcontaining mixed chain-length alkanes into sebacic acid in a partiallybeta-oxidation blocked microorganism. The mixed chain-length alkanes arefirst converted into mixed chain-length fatty acids and then mixeddiacids by activities in the omega-oxidation pathway. Mixed diacids canbe converted to sebacic acid by activities in the beta-oxidationpathway, with the simultaneous generation of acetyl-CoA.

FIG. 8 is a schematic representation of the conversion of a feedstockcontaining mixed chain-length alkanes into dodecanedioic acid in apartially beta-oxidation blocked microorganism. The mixed chain-lengthalkanes are first converted into mixed chain-length fatty acids and thenmixed diacids by activities in the omega-oxidation pathway. Mixeddiacids can be converted to dodecanedioic acid by activities in thebeta-oxidation pathway, with the simultaneous generation of acetyl-CoA.

FIG. 9 graphically illustrates the conversion of decane to sebacic acidin a fully beta-oxidation blocked C. tropicalis yeast strain. Afterincubation for the times shown in the graph, the media was subjected togas chromatography. The results indicate that greater than 99% of thedecane was converted into sebacic acid, with a minimal amount of capricacid also detected by gas chromatography. No significant accumulation ofany other monoacid or diacid was detected by gas chromatography.Experimental details and results are given in Example 1.

FIG. 10 graphically illustrates the conversion of capric acid to sebacicacid in a C. tropicalis yeast strain. GC analysis was performed after apredetermined period of growth. Nearly all the capric acid added wasconverted to sebacic acid using a starting concentration of capric acid.Experimental details and results are given in Example 2.

FIG. 11 graphically illustrates the distribution of diacids producedduring the conversion of long-chain fatty acids to mixed diacids underfermentation conditions using a partially beta-oxidation blocked Candidatropicalis strain (e.g., sAA106). Experimental details and results aregiven in Example 5.

FIG. 12 graphically illustrates the conversion of decane to sebacic acidin a fully beta-oxidation blocked C. tropicalis yeast strain havingadditional genetic modifications. Strain sAA003 is the fullybeta-oxidation blocked control strain. +CPR indicates the fullybeta-oxidation blocked strain also includes an increased number ofcopies of cytochrome P450 reductase. +CPR+A12 indicates starting strainsAA003 includes the addition genetic modifications of an increasednumber of copies of cytochrome P450 reductase and also includes anincreased number of copies of cytochrome P450 A12 (e.g., CYP52A12).+CPR+A18 indicates starting strain sAA003 includes the addition geneticmodifications of an increased number of copies of cytochrome P450reductase and also includes an increased number of copies of cytochromeP450 A18 (e.g., CYP52A18). +CPR+A19 indicates starting strain sAA003includes the addition genetic modifications of an increased number ofcopies of cytochrome P450 reductase and also includes an increasednumber of copies of cytochrome P450 A19 (e.g., CYP52A19). +CPR+A20indicates starting strain sAA003 includes the addition geneticmodifications of an increased number of copies of cytochrome P450reductase and also includes an increased number of copies of cytochromeP450 A20 (e.g., CYP52A20). Experimental details and results are given inExample 7.

FIG. 13 graphically illustrates the results of conversion of methyllaurate to dodecanedioic acid in a fully beta-oxidation blocked C.tropicalis yeast strain also contain genetic alterations to amonooxygenase reductase activity, a monooxygenase activity, or amonooxygenase reductase activity and a monooxygenase activity. After 48hours of incubation the media was subjected to gas chromatography. Theresults indicate that Candida strains containing an increased number ofcopies of a CYP52A18 monooxygenase activity and an increased number ofcopies of a monooxygenase reductase activity (e.g., CPR750) gave thehighest yield of dodecanedioic acid (e.g., DDDA), in shake flaskfermentation experiments. Experimental details and results are given inExample 8.

FIG. 14 and FIG. 15 schematically illustrate a screening and/orselection method for identifying acyl-CoA oxidase activities withspecific substrate specificities. The method can be utilized inconjunction with generating and/or identifying acyl-CoA oxidaseactivities with altered chain-length substrate specificities.Screening/selection method details are given in Example 9.

FIG. 16 graphically illustrates the results of engineered microorganismsdescribed herein converting decane to sebacic acid under fermentationconditions using different amounts of decane as the feedstock.Experimental details and results are given in Example 3.

FIG. 17 graphically illustrates the results of engineered microorganismsdescribed herein converting a mixed fatty acid feedstock (e.g., mixedchain-length fatty acids) to sebacic acid under fermentation conditions.Experimental details and results are given in Example 4.

FIG. 18 shows a diagram of a plasmid designated pAA073 containing a POX4promoter and a POX4 terminator.

FIG. 19 shows a diagram of a plasmid designated pAA298.

FIG. 20 shows the production of either dodecanedioic acid from methyllaurate (ML) or tetradecanedioic acid from methyl myristate (MM)utilizing strains sAA1306 and sAA003.

FIG. 21 shows the production of either dodecanedioic acid from methyllaurate (ML) or tetradecanedioic acid from methyl myristate (MM) usingstrains sAA1082 and sAA003.

FIG. 22 shows the production cis-9-octadecenedioic acid (C18:1 diacid)from oleic acid for four fully beta-oxidation blocked strains. The datapoints are derived from the averages of three identical fermentations.

FIG. 23 shows the concentrations of HFAs produced during the omegaoxidation of oleic acid by strains sAA003, sAA1233, sAA1306 and sAA1485.

FIG. 24 shows the production of decanedioic acid (sebacic acid) andcompares the productivity of the two strains under the two differentinduction conditions.

FIG. 25 shows the amount of decanoic acid produced under the differentfermentation conditions.

FIG. 26 shows the production of DDDA and 12-hydroxy-dodecanoic acid(HFA) from methyl laurate.

FIG. 27 shows the production of DDDA from methyl laurate.

FIG. 28 shows the production of DDDA.

FIG. 29 shows the production of HFAs from methyl laurate.

FIG. 30 shows a promoter replacement strategy.

FIG. 31 shows an example of the production of HFAs during the firstoxidation step in the omega-oxidation pathway.

DETAILED DESCRIPTION

Certain fatty dicarboxylic acids (i.e., diacids, e.g., dodecanedioicacid or sebacic acid) are chemical intermediates in manufacturingprocesses used to make certain polyamides, polyurethanes andplasticizers, all of which have wide applications in producing itemssuch as antiseptics, top-grade coatings, hot-melt coating and adhesives,painting materials, corrosion inhibitor, surfactant, engineeringplastics and can also be used as a starting material in the manufactureof fragrances, for example. For example dodecanedioic acid, also knownas 1,12 dodecanedioic acid, and DDDA, is a 12 carbon organic moleculethat is a fatty dicarboxylic acid. In another example, sebacic acid,also known as 1,10 decanedioic acid, and 1,8 octanedicarboxylic acid, isa 10 carbon organic molecule that is a fatty dicarboxylic acid.

Provided herein are methods for producing a fatty dicarboxylic acid(also referred to herein as a diacid). Any suitable diacid can beproduced, and a diacid produced often includes acid moieties at eachterminus of the molecule (e.g., alpha omega diacids). A diacid sometimesis a C4 to a C24 diacid (i.e., a diacid containing 4 carbons to 24carbons) and sometimes is a C8, C10, C12, C14, C16, C18, or C20 diacid.Yeast and processes herein are capable of producing a diacid containingan odd number of carbons, and sometimes a product contains one or morediacids chosen from a C5, C7, C9, C11, C13, C15, C17, C19, C21 and C23diacid. A hydrocarbon portion of a diacid sometimes is fully saturatedand sometimes a diacid includes one or more unsaturations (e.g., doublebonds).

Non-limiting examples of diacids include octanedioic acid, decanedioicacid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) and other organicintermediates using biological systems. Non-limiting examples of fattydicarboxylic acids include suberic acid (i.e., octanedioic acid,1,8-octanedioic acid, octanedioic acid, octane-1,8-dioic acid,1,6-hexanedicarboxylic acid, capryllic diacids), sebacic acid (i.e.,1,10-decanedioic acid, decanedioic acid, decane-1,10-dioic acid,1,8-octanedicarboxylic acid, capric diacid), dodecanedioic acid (i.e.,DDDA, 1,12-dodecanedioic acid, dodecanedioic acid, dodecane-1,12-dioicacid, 1,10-decanedicarboxylic acid, decamethylenedicaboxylic acid,1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e., TDDA,1,14-tetradecanedioic acid, tetradecanedioic acid,tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid, myristicdiacid), thapsic acid (i.e., hexadecanedioic acid, 1,16-hexadecanedioicacid, hexadecanedioic acid, hexadecane-1,16-dioic acid,1,14-tetradecanedicarboxylic acid, palmitic diacid),cis-9-hexadecenedioic acid (i.e., palmitoleic diacids), octadecanedioicacid (i.e., 1,18-octadecanedioic acid, octadecanedioic acid,octadecane-1,18-dioic acid, 1,16-hexadecanedicarboxylic acid, stearicdiacid), cis-9-octadecenedioic acid (i.e., oleic diacids),cis-9,12-octadecenedioic acid (i.e., linoleic diacids),cis-9,12,15-octadecenedioic acid (i.e., linolenic diacids), arachidicdiacid (i.e., eicosanoic diacid, icosanoic diacid), 11-eicosenoic diacid(i.e., cis-11-eicosenedioic acid), 13-eicosenoic diacids (i.e.,cis-13-eicosenedioic acid), arachidonic diacid (i.e.,cis-5,8,11,14-eicosatetraenedioic acid).

A genetically modified yeast can be provided with a feedstock to producea diacid, and the feedstock sometimes includes a substantially purealiphatic molecule from which the diacid is produced. In certainembodiments, the feedstock contains a mixed set of aliphatic moleculesfrom which diacids may be produced. In some embodiments, an aliphaticmolecule in the feedstock is the predominant aliphatic species andsometimes a particular diacid produced from that aliphatic molecule isthe predominant diacid species produced. A predominant species generallyis 51% or more by weight of aliphatic molecule species in a feedstock or51% or more by weight of diacid species in a product (e.g., about 55% ormore, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more,85% or more, 90% or more or 95% or more).

Such production systems may have significantly less environmental impactand could be economically competitive with current manufacturingsystems. Thus, provided in part herein are methods for manufacturing afatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) by engineered microorganisms.In some embodiments microorganisms are engineered to contain at leastone heterologous gene encoding an enzyme, where the enzyme is a memberof a novel and/or altered pathway engineered into the microorganism. Incertain embodiments, an organism may be selected for elevated activityof a native enzyme.

Microorganisms

A microorganism selected often is suitable for genetic manipulation andoften can be cultured at cell densities useful for industrial productionof a target fatty dicarboxylic acid product. A microorganism selectedoften can be maintained in a fermentation device.

The term “engineered microorganism” as used herein refers to a modifiedmicroorganism that includes one or more activities distinct from anactivity present in a microorganism utilized as a starting point(hereafter a “host microorganism”). An engineered microorganism includesa heterologous polynucleotide in some embodiments, and in certainembodiments, an engineered organism has been subjected to selectiveconditions that alter an activity, or introduce an activity, relative tothe host microorganism. Thus, an engineered microorganism has beenaltered directly or indirectly by a human being. A host microorganismsometimes is a native microorganism, and at times is a microorganismthat has been engineered to a certain point.

In some embodiments an engineered microorganism is a single cellorganism, often capable of dividing and proliferating. A microorganismcan include one or more of the following features: aerobe, anaerobe,filamentous, non-filamentous, monoploid, dipoid, auxotrophic and/ornon-auxotrophic. In certain embodiments, an engineered microorganism isa prokaryotic microorganism (e.g., bacterium), and in certainembodiments, an engineered microorganism is a non-prokaryoticmicroorganism. In some embodiments, an engineered microorganism is aeukaryotic microorganism (e.g., yeast, fungi, amoeba). In someembodiments, an engineered microorganism is a fungus. In someembodiments, an engineered organism is a yeast.

Any suitable yeast may be selected as a host microorganism, engineeredmicroorganism, genetically modified organism or source for aheterologous or modified polynucleotide. Yeast include, but are notlimited to, Yarrowia yeast (e.g., Y. lipolytica (formerly classified asCandida lipolytica)), Candida yeast (e.g., C. revkaufi, C. viswanathii,C. pulcherrima, C. tropicalis, C. utilis), Rhodotorula yeast (e.g., R.glutinus, R. graminis), Rhodosporidium yeast (e.g., R. toruloides),Saccharomyces yeast (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S.carlsbergensis), Cryptococcus yeast, Trichosporon yeast (e.g., T.pullans, T. cutaneum), Pichia yeast (e.g., P. pastoris) and Lipomycesyeast (e.g., L. starkeyii, L. lipoferus). In some embodiments, asuitable yeast is of the genus Arachniotus, Aspergillus, Aureobasidium,Auxarthron, Blastomyces, Candida, Chrysosporuim, ChrysosporuimDebaryomyces, Coccidiodes, Cryptococcus, Gymnoascus, Hansenula,Histoplasma, Issatchenkia, Kluyveromyces, Lipomyces, Lssatchenkia,Microsporum, Myxotrichum, Myxozyma, Oidiodendron, Pachysolen,Penicillium, Pichia, Rhodosporidium, Rhodotorula, Rhodotorula,Saccharomyces, Schizosaccharomyces, Scopulariopsis, Sepedonium,Trichosporon, or Yarrowia. In some embodiments, a suitable yeast is ofthe species Arachniotus flavoluteus, Aspergillus flavus, Aspergillusfumigatus, Aspergillus niger, Aureobasidium pullulans, Auxarthronthaxteri, Blastomyces dermatitidis, Candida albicans, Candidadubliniensis, Candida famata, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica,Candida lustitaniae, Candida parapsilosis, Candida pulcherrima, Candidarevkaufi, Candida rugosa, Candida tropicalis, Candida utilis, Candidaviswanathii, Candida xestobii, Chrysosporuim keratinophilum, Coccidiodesimmitis, Cryptococcus albidus var. diffluens, Cryptococcus laurentii,Cryptococcus neofomans, Debaryomyces hansenii, Gymnoascus dugwayensis,Hansenula anomala, Histoplasma capsulatum, Issatchenkia occidentalis,Isstachenkia orientalis, Kluyveromyces lactis, Kluyveromyces marxianus,Kluyveromyces thermotolerans, Kluyveromyces waltii, Lipomyces lipoferus,Lipomyces starkeyii, Microsporum gypseum, Myxotrichum deflexum,Oidiodendron echinulatum, Pachysolen tannophilis, Penicillium notatum,Pichia anomala, Pichia pastoris, Pichia stipitis, Rhodosporidiumtoruloides, Rhodotorula glutinus, Rhodotorula graminis, Saccharomycescerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe,Scopulariopsis acremonium, Sepedonium chrysospermum, Trichosporoncutaneum, Trichosporon pullans, Yarrowia lipolytica, or Yarrowialipolytica (formerly classified as Candida lipolytica). In someembodiments, a yeast is a Y. lipolytica strain that includes, but is notlimited to, ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982 andLGAM S(7)1 strains (Papanikolaou S., and Aggelis G., Bioresour. Technol.82(1):43-9 (2002)). In certain embodiments, a yeast is a Candida species(i.e., Candida spp.) yeast. Any suitable Candida species can be usedand/or genetically modified for production of a fatty dicarboxylic acid(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid). In some embodiments, suitable Candida speciesinclude, but are not limited to Candida albicans, Candida dubliniensis,Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr,Candida krusei, Candida lambica, Candida lipolytica, Candidalustitaniae, Candida parapsilosis, Candida pulcherrima, Candidarevkaufi, Candida rugosa, Candida tropicalis, Candida utilis, Candidaviswanathii, Candida xestobii and any other Candida spp. yeast describedherein. Non-limiting examples of Candida spp. strains include, but arenot limited to, sAA001 (ATCC20336), sAA002 (ATCC20913), sAA003(ATCC20962), sAA496 (US2012/0077252), sAA106 (US2012/0077252), SU-2(ura3-/ura3-), H5343 (beta oxidation blocked; U.S. Pat. No. 5,648,247)strains. Any suitable strains from Candida spp. yeast may be utilized asparental strains for genetic modification.

Yeast genera, species and strains are often so closely related ingenetic content that they can be difficult to distinguish, classifyand/or name. In some cases strains of C. lipolytica and Y. lipolyticacan be difficult to distinguish, classify and/or name and can be, insome cases, considered the same organism. In some cases, various strainsof C.tropicalis and C. viswanathii can be difficult to distinguish,classify and/or name (for example see Arie et. al., J. Gen. Appl.Microbiol., 46, 257-262 (2000). Some C. tropicalis and C. viswanathiistrains obtained from ATCC as well as from other commercial or academicsources can be considered equivalent and equally suitable for theembodiments described herein. In some embodiments, some parental stainsof C.tropicalis and C. viswanathii are considered to differ in nameonly.

Any suitable fungus may be selected as a host microorganism, engineeredmicroorganism or source for a heterologous polynucleotide. Non-limitingexamples of fungi include, but are not limited to, Aspergillus fungi(e.g., A. parasiticus, A. nidulans), Thraustochytrium fungi,Schizochytrium fungi and Rhizopus fungi (e.g., R. arrhizus, R. oryzae,R. nigricans). In some embodiments, a fungus is an A. parasiticus strainthat includes, but is not limited to, strain ATCC24690, and in certainembodiments, a fungus is an A. nidulans strain that includes, but is notlimited to, strain ATCC38163.

Any suitable prokaryote may be selected as a host microorganism,engineered microorganism or source for a heterologous polynucleotide. AGram negative or Gram positive bacteria may be selected. Examples ofbacteria include, but are not limited to, Bacillus bacteria (e.g., B.subtilis, B. megaterium), Acinetobacter bacteria, Norcardia baceteria,Xanthobacter bacteria, Escherichia bacteria (e.g., E. coli (e.g.,strains DH10B, Stb12, DH5-alpha, DB3, DB3.1), DB4, DB5, JDP682 andccdA-over (e.g., U.S. application Ser. No. 09/518,188)), Streptomycesbacteria, Erwinia bacteria, Klebsiella bacteria, Serratia bacteria(e.g., S. marcessans), Pseudomonas bacteria (e.g., P. aeruginosa),Salmonella bacteria (e.g., S. typhimurium, S. typhi), Megasphaerabacteria (e.g., Megasphaera elsdenii). Bacteria also include, but arenot limited to, photosynthetic bacteria (e.g., green non-sulfurbacteria, Choroflexus bacteria (e.g., C. aurantiacus), Chloronemabacteria (e.g., C. gigateum)), green sulfur bacteria (e.g., Chlorobiumbacteria (e.g., C. limicola)), Pelodictyon bacteria (e.g., P. luteolum),purple sulfur bacteria (e.g., Chromatium bacteria (e.g., C. okenii)),and purple non-sulfur bacteria (e.g., Rhodospirillum bacteria (e.g., R.rubrum), Rhodobacter bacteria (e.g., R. sphaeroides, R. capsulatus), andRhodomicrobium bacteria (e.g., R. vanellii)).

Cells from non-microbial organisms can be utilized as a hostmicroorganism, engineered microorganism or source for a heterologouspolynucleotide. Examples of such cells, include, but are not limited to,insect cells (e.g., Drosophila (e.g., D. melanogaster), Spodoptera(e.g., S. frugiperda Sf9 or Sf21 cells) and Trichoplusa (e.g., High-Fivecells); nematode cells (e.g., C. elegans cells); avian cells; amphibiancells (e.g., Xenopus laevis cells); reptilian cells; mammalian cells(e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanomaand HeLa cells); and plant cells (e.g., Arabidopsis thaliana, Nicotaniatabacum, Cuphea acinifolia, Cuphea aequipetala, Cuphea angustifolia,Cuphea appendiculata, Cuphea avigera, Cuphea avigera var. pulcherrima,Cuphea axilliflora, Cuphea bahiensis, Cuphea baillonis, Cupheabrachypoda, Cuphea bustamanta, Cuphea calcarata, Cuphea calophylla,Cuphea calophylla subsp. mesostemon, Cuphea carthagenensis, Cupheacircaeoides, Cuphea confertiflora, Cuphea cordata, Cuphea crassiflora,Cuphea cyanea, Cuphea decandra, Cuphea denticulata, Cuphea disperma,Cuphea epilobiifolia, Cuphea ericoides, Cuphea flava, Cupheaflavisetula, Cuphea fuchsiifolia, Cuphea gaumeri, Cuphea glutinosa,Cuphea heterophylla, Cuphea hookeriana, Cuphea hyssopifolia(Mexican-heather), Cuphea hyssopoides, Cuphea ignea, Cuphea ingrata,Cuphea jorullensis, Cuphea lanceolata, Cuphea linarioides, CupheaIlavea, Cuphea lophostoma, Cuphea lutea, Cuphea lutescens, Cupheamelanium, Cuphea melvilla, Cuphea micrantha, Cuphea micropetala, Cupheamimuloides, Cuphea nitidula, Cuphea palustris, Cuphea parsonsia, Cupheapascuorum, Cuphea paucipetala, Cuphea procumbens, Cuphea pseudosilene,Cuphea pseudovaccinium, Cuphea pulchra, Cuphea racemosa, Cuphea repens,Cuphea salicifolia, Cuphea salvadorensis, Cuphea schumannii, Cupheasessiliflora, Cuphea sessilifolia, Cuphea setosa, Cuphea spectabilis,Cuphea spermacoce, Cuphea splendida, Cuphea splendida var. viridiflava,Cuphea strigulosa, Cuphea subuligera, Cuphea teleandra, Cupheathymoides, Cuphea tolucana, Cuphea urens, Cuphea utriculosa, Cupheaviscosissima, Cuphea watsoniana, Cuphea wrightii, Cuphea lanceolata)).

Microorganisms or cells used as host organisms or source for aheterologous polynucleotide are commercially available. Microorganismsand cells described herein, and other suitable microorganisms and cellsare available, for example, from Invitrogen Corporation (Carlsbad,Calif.), American Type Culture Collection (Manassas, Va.), andAgricultural Research Culture Collection (NRRL; Peoria, Ill.).

Host microorganisms and engineered microorganisms may be provided in anysuitable form. For example, such microorganisms may be provided inliquid culture or solid culture (e.g., agar-based medium), which may bea primary culture or may have been passaged (e.g., diluted and cultured)one or more times. Microorganisms also may be provided in frozen form ordry form (e.g., lyophilized). Microorganisms may be provided at anysuitable concentration.

Carbon Processing Pathways and Activities

FIGS. 1-8 schematically illustrate non-limiting embodiments ofengineered pathways that can be used to produce a fatty dicarboxylicacid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid) from various starting carbon sources or feedstocks.FIG. 1 depicts an embodiment of a non-limiting engineered biologicalpathway for producing sebacic acid in microorganisms having a fullyblocked beta-oxidation pathway, using decane as the carbon sourcestarting material. FIG. 2 depicts an embodiment of a non-limitingengineered biological pathway for producing dodecanedioic acid inmicroorganisms having a fully blocked beta-oxidation pathway, usingdodecane as the carbon source starting material. FIG. 3 and FIG. 4depict an embodiment of a non-limiting engineered biological pathway forproducing mixed chain-length diacids in a microorganism having a fullyblocked beta-oxidation pathway, using mixed chain-length alkanes as thecarbon source starting material. Sebacic acid (FIG. 3) and dodecanedioicacid (FIG. 4) can be separated and/or purified away from other diacidproducts using a suitable combination of centrifugation, organic solventextraction, chromatography, and/or other purification/separationtechniques. FIG. 5 and FIG. 6 depict an embodiment of a non-limitingengineered biological pathway for producing sebacic acid (FIG. 5) anddodecanedioic acid (FIG. 6) in microorganisms having a partially blockedbeta oxidation pathway, using long-chain alkanes as the carbon sourcestarting material. FIG. 7 and FIG. 8 depict an embodiment of anon-limiting engineered biological pathway for producing sebacic acid(FIG. 7) and dodecanedioic acid (FIG. 8) in microorganisms having apartially blocked beta oxidation pathway, using mixed-chain lengthalkanes as the carbon source starting material. The alkane carbon sourcestarting materials are initially metabolized using naturally occurringand/or engineered activities in naturally occurring and/or engineeredpathways to yield an intermediate alcohol which can then be converted toa carboxylic acid (e.g., fatty acid) by the action of other naturallyoccurring and/or engineered activities in the omega-oxidation pathwaydepicted in FIGS. 1-8.

Alkanes are omega-hydroxylated by the activity of cytochrome P450enzymes, thereby generating the equivalent chain-length alcoholderivative of the starting alkane carbon source material. In certainembodiments, a cytochrome P450 activity can be increased by increasingthe number of copies of a cytochrome P450 gene (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25 or more copies of the gene), by increasing theactivity of a promoter that regulates transcription of a cytochrome P450gene, or by increasing the number of copies of a cytochrome P450 geneand increasing the activity of a promoter that regulates transcriptionof a cytochrome P450 gene, thereby increasing the production of targetproduct (e.g., sebacic or dodecanedioic acid) via increased activity ofone or more cytochrome P450 enzymes. In some embodiments, a cytochromeP450 enzyme is endogenous to the host microorganism. One or morecytochrome P450 activities can be added and/or increased dependent onthe carbon source starting material, in certain embodiments. CytochromeP450's sometimes exhibit increased activities in response to stimulationby certain feedstocks or carbon source starting materials. In someembodiments, an engineered microorganism includes an increased number ofcopies of one or more cytochrome P450s that are stimulated by a chosencarbon source starting material or feedstock. Cytochrome P450responsiveness to a chosen starting carbon source or feedstock can bedetermined using any suitable assay. Non-limiting examples of assayssuitable for identification of cytochrome P450 responsiveness to astarting carbon source or feedstock include RT-PCR or qRT-PCR after thehost microorganism has been exposed to the chosen carbon source orfeedstock for varying amounts of time.

Cytochrome P450 is reduced by the activity of cytochrome P450 reductase(CPR), thereby recycling cytochrome P450 to allow further enzymaticactivity. In certain embodiments, the CPR enzyme is endogenous to thehost microorganism. In some embodiments, host CPR activity can beincreased by increasing the number of copies of a CPR gene (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more copies of the gene), byincreasing the activity of a promoter that regulates transcription of aCPR gene, or by increasing the number of copies of a CPR gene andincreasing the activity of a promoter that regulates transcription of aCPR gene, thereby increasing the production of target product (e.g.,sebacic or dodecanedioic acid) via increased recycling of cytochromeP450. In certain embodiments, the promoter can be a heterologouspromoter (e.g., endogenous or exogenous promoter). In some embodiments,the CPR gene is heterologous and exogenous and can be isolated from anysuitable organism. Non-limiting examples of organisms from which a CPRgene can be isolated include C. tropicalis, S. cerevisiae and Bacillusmegaterium.

Oxidation of the alcohol to an aldehyde may be performed by an enzyme inthe fatty alcohol oxidase family (e.g., long-chain fatty alcohol oxidaseEC 1.1.3.20), or an enzyme in the alcohol dehydrogenase family (e.g.,fatty alcohol dehydrogenase; EC 1.1.1.1). The aldehyde may be oxidizedto a carboxylic acid (e.g., sebacic or dodecanedioic acid) by theactivity of the enzyme aldehyde dehydrogenase (e.g., long-chain-aldehydedehydrogenase or fatty aldehyde dehydrogenase; EC 1.2.1.48). In someembodiments, the long chain fatty alcohol oxidase, fatty alcoholdehydrogenase and/or the long-chain-aldehyde dehydrogenase exist in ahost organism. Flux through these two steps may sometimes be augmentedby increasing the copy number of the enzymes, or by increasing theactivity of the promoter transcribing the genes. In some embodimentsalcohol and aldehyde dehydrogenases specific for 10, 12 or 14 carbonsubstrates may be isolated from another organism, and inserted into thehost organism.

FIG. 1 depicts a non-limiting embodiment of an engineered biologicalpathway for making sebacic acid using decane (e.g., a C10 alkane) as thecarbon source starting material. Due to the carbon chain length ofdecane, no chain shortening is necessary to arrive at the 10 carbondiacid, sebacic acid. Thus a fully beta oxidation blocked microorganismcan be utilized to minimize conversion of the desired 10 carbon diacidinto diacids having shorter chain lengths.

FIG. 2 depicts a non-limiting embodiment of an engineered biologicalpathway for making dodecanedioic acid using dodecane (e.g., a C12alkane) as the carbon source starting material. Due to the carbon chainlength of dodecane, no chain shortening is necessary to arrive at the 12carbon diacid, dodecanedioic acid. Thus a fully beta oxidation blockedmicroorganism can be utilized to minimize conversion of the desired 12carbon diacid into diacids having shorter chain lengths.

FIGS. 3 and 4 depict a non-limiting embodiment of an engineeredbiological pathway for generating a mixed population of diacid (fattydicarboxylic acid) products, including sebacid acid (FIG. 3) anddodecanedioic acid (FIG. 4), using a carbon source or feedstock thatcontains mixed-chain-length alkanes as the carbon source startingmaterial. Any suitable mixed-chain-length alkane, fatty alcohol, mixedchain length fatty alcohol feedstock, fatty acid, mixed fatty acidfeedstock, paraffin, fat or oil can be used. In some embodiments, thedistribution of carbon chain lengths in the starting material issubstantially similar to the desired carbon chain length distribution inthe mixed diacid product. In certain embodiments, the feedstock isenriched for a desired chain length. In some embodiments, the enrichedfraction is enriched for carbon chain lengths of about 10 carbons. Insome embodiments, the enriched fraction is enriched for carbon chainlengths of about 12 carbons. Because, in some embodiments, the diacidsgenerated have substantially the same chain lengths as the chain lengthsfound in the carbon source starting material, a fully beta-oxidationblocked microorganism can be utilized to minimize conversion of thediacids of desired chain length into diacids of shorter chain lengths.The lower part of the pathways in FIG. 3 and FIG. 4 show the separationof sebacic acid and dodecanedioic acid, respectively, away from themixed diacid products by the use of separation techniques describedherein, or those known in the art.

In certain embodiments involving genetically modified organisms havingpartially blocked beta-oxidation pathways (see FIGS. 5-8), feedstockssuitable for use include, but are not limited to, fatty acid distillatesor soapstocks of renewable oils (palm oil fatty acid distillate, soybeanoil soapstock, coconut oil soapstock), renewable oils (coconut oil, palmoil, palm kernel oil, soybean oil, corn oil, etc.), fatty acids of chainlength equal to or greater than C10 (in substantially single form (e.g.,in substantially pure form) or in mixture form, alkanes of chain lengthequal to or greater than C10 in substantially single form (e.g.,substantially pure form) or in mixture form.

Carbon sources with longer chain lengths (e.g., 12 carbons or greater inlength) can be metabolized using naturally occurring and/or engineeredpathways to yield molecules that can be further metabolized using thebeta oxidation pathway shown in the lower portion of FIGS. 5-8. In someembodiments, beta-oxidation activities in the pathways shown in FIGS.5-8 also can be engineered (e.g., as described herein) to enhancemetabolism and target product formation. In some embodiments, oneacyl-CoA oxidase activity of the beta-oxidation pathway is engineered tobe enhanced, and in certain embodiments, the other acyl-CoA oxidaseactivity in the beta-oxidation pathway is altered to reduce or eliminatethe activity, thereby optimizing the production of a diacid of a desiredchain-length or diacids with a distribution of desired chain lengths. Insome embodiments, an acyl-CoA oxidase is selected and/or engineered toalter the substrate specificity of the enzyme. In certain embodiments,the substrate specificity of a heterologous and/or engineered acyl-CoAoxidase is for carbon chain lengths of between about 12 carbons andabout 18 carbons, and in some embodiments a heterologous and/orengineered acyl-CoA oxidase exhibits no activity on substrates below 12carbons in length. In certain embodiments, a heterologous acyl-CoAoxidase with a desired chain length specificity can be isolated from anysuitable organism. Non-limiting examples of organisms that include, orcan be used as donors for acyl-CoA oxidase enzymes include yeast (e.g.,Candida, Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).

In certain embodiments, a carbon source starting material (e.g., alkane,fatty acid, fatty alcohol, dicarboxylic acid) of intermediate or longchain length (e.g., between about 10 carbons and 22 carbons) isconverted into an acyl-CoA derivative for entry into the beta-oxidationpathway. The acyl-CoA derivative can be generated by the activity of anacyl-CoA ligase enzyme, in some embodiments. The acyl-CoA derivative issubsequently oxidized by the activity of an acyl-CoA oxidase enzyme(e.g., also known as acyl-CoA oxidoreductase and fatty acyl-coenzyme Aoxidase) of natural or altered substrate specificity, in certainembodiments. The trans-2,3-dehydroacyl-CoA derivative long chain fattyalcohol, fatty acid or dicarboxylic acid may be further converted to3-hydroxyacyl-CoA by the activity of enoyl-CoA hydratase.3-hydroxyacyl-CoA can be converted to 3-oxoacyl-CoA by the activity of3-hydroxyacyl-CoA dehydrogenase. 3-oxoacyl-CoA may be converted to anacyl-CoA molecule, shortened by 2 carbons and an acetyl-CoA, by theactivity of Acetyl-CoA C-acyltransferase (e.g., also known asbeta-ketothiolase and beta-ketothiolase). In some embodiments, acyl-CoAmolecules may be repeatedly shortened by beta oxidation until a desiredcarbon chain length is generated (e.g., 10 or 12 carbons, sebacic acidor dodecanedioic acid, respectively). A shortened fatty acid can befurther processed using omega oxidation to yield a dicarboxylic acid(e.g., dodecanedioic acid).

Beta-Oxidation Activities

The term “beta oxidation pathway” as used herein, refers to a series ofenzymatic activities utilized to metabolize fatty alcohols, fatty acids,or dicarboxylic acids. The activities utilized to metabolize fattyalcohols, fatty acids, or dicarboxylic acids include, but are notlimited to, acyl-CoA ligase activity, acyl-CoA oxidase activity,acyl-CoA hydrolase activity, acyl-CoA thioesterase activity, enoyl-CoAhydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity andacetyl-CoA C-acyltransferase activity. The term “beta oxidationactivity” refers to any of the activities in the beta oxidation pathwayutilized to metabolize fatty alcohols, fatty acids or dicarboxylicacids.

Beta-Oxidation—Acyl-CoA Ligase

An acyl-CoA ligase enzyme sometimes is encoded by the host organism andcan be added to generate an engineered organism. In some embodiments,host acyl-CoA ligase activity can be increased by increasing the numberof copies of an acyl-CoA ligase gene, by increasing the activity of apromoter that regulates transcription of an acyl-CoA ligase gene, or byincreasing the number copies of the gene and by increasing the activityof a promoter that regulates transcription of the gene, therebyincreasing production of target product (e.g., sebacic or dodecanedioicacid) due to increased carbon flux through the pathway. In certainembodiments, the acyl-CoA ligase gene can be isolated from any suitableorganism. Non-limiting examples of organisms that include, or can beused as donors for, acyl-CoA ligase enzymes include Candida,Saccharomyces, or Yarrowia.

Beta-Oxidation—Enoyl-CoA Hydratase

An enoyl-CoA hydratase enzyme catalyzes the addition of a hydroxyl groupand a proton to the unsaturated β-carbon on a fatty-acyl CoA andsometimes is encoded by the host organism and sometimes can be added togenerate an engineered organism. In certain embodiments, the enoyl-CoAhydratase activity is unchanged in a host or engineered organism. Insome embodiments, the host enoyl-CoA hydratase activity can be increasedby increasing the number of copies of an enoyl-CoA hydratase gene, byincreasing the activity of a promoter that regulates transcription of anenoyl-CoA hydratase gene, or by increasing the number copies of the geneand by increasing the activity of a promoter that regulatestranscription of the gene, thereby increasing the production of targetproduct (e.g., sebacic or dodecanedioic acid) due to increased carbonflux through the pathway. In certain embodiments, the enoyl-CoAhydratase gene can be isolated from any suitable organism. Non-limitingexamples of organisms that include, or can be used as donors for,enoyl-CoA hydratase enzymes include Candida, Saccharomyces, or Yarrowia.

Beta-Oxidation—3-Hydroxyacyl-CoA Dehydrogenase

3-hydroxyacyl-CoA dehydrogenase enzyme catalyzes the formation of a3-ketoacyl-CoA by removal of a hydrogen from the newly formed hydroxylgroup created by the activity of enoyl-CoA hydratase. In someembodiments, the activity is encoded by the host organism and sometimescan be added or increased to generate an engineered organism. In certainembodiments, the 3-hydroxyacyl-CoA activity is unchanged in a host orengineered organism. In some embodiments, the host 3-hydroxyacyl-CoAdehydrogenase activity can be increased by increasing the number ofcopies of a 3-hydroxyacyl-CoA dehydrogenase gene, by increasing theactivity of a promoter that regulates transcription of a3-hydroxyacyl-CoA dehydrogenase gene, or by increasing the number copiesof the gene and by increasing the activity of a promoter that regulatestranscription of the gene, thereby increasing production of targetproduct (e.g., sebacic or dodecanedioic acid) due to increased carbonflux through the pathway. In certain embodiments, the 3-hydroxyacyl-CoAdehydrogenase gene can be isolated from any suitable organism.Non-limiting examples of organisms that include, or can be used asdonors for, 3-hydroxyacyl-CoA dehydrogenase enzymes include Candida,Saccharomyces, or Yarrowia.

Beta-Oxidation—Acetyl-CoA C-Acyltransferase

An Acetyl-CoA C-acyltransferase (e.g., beta-ketothiolase) enzymecatalyzes the formation of a fatty acyl-CoA shortened by 2 carbons bycleavage of the 3-ketoacyl-CoA by the thiol group of another molecule ofCoA. The thiol is inserted between C-2 and C-3, which yields an acetylCoA molecule and an acyl CoA molecule that is two carbons shorter. AnAcetyl-CoA C-acyltransferase sometimes is encoded by the host organismand sometimes can be added to generate an engineered organism. Incertain embodiments, the acetyl-CoA C-acyltransferase activity isunchanged in a host or engineered organism. In some embodiments, thehost acetyl-CoA C-acyltransferase activity can be increased byincreasing the number of copies of an acetyl-CoA C-acyltransferase gene,or by increasing the activity of a promoter that regulates transcriptionof an acetyl-CoA C-acyltransferase gene, thereby increasing theproduction of target product (e.g., sebacic or dodecanedioic acid) dueto increased carbon flux through the pathway. In certain embodiments,the acetyl-CoA C-acyltransferase gene can be isolated from any suitableorganism. Non-limiting examples of organisms that include, or can beused as donors for, acetyl-CoA C-acyltransferase enzymes includeCandida, Saccharomyces, or Yarrowia.

Omega Oxidation Activities

Targets for improving the productivity of diacid product formation fromfatty acid feedstocks in β-oxidation blocked strains are often thosewhich can improve carbon flux through the w-oxidation pathway. In someembodiments, these targets are: 1) enzymes performing the rate-limitingstep in the w-oxidation pathway (e.g., CPR and CYP450), 2) enzymesperforming fatty acid transport into the cell (e.g., Acyl CoASynthetases), and 3) enzymes that provide the cofactors required for thew-oxidation pathway (e.g., G6PDH).

The term “omega oxidation activity” refers to any of the activities inthe omega oxidation pathway utilized to metabolize alkanes, fattyalcohols, fatty acids, dicarboxylic acids, or sugars. The activitiesutilized to metabolize fatty alcohols, fatty acids, or dicarboxylicacids include, but are not limited to, monooxygenase activity (e.g.,cytochrome P450 activity), monooxygenase reductase activity (e.g.,cytochrome P450 reductase activity), alcohol dehydrogenase activity(e.g., fatty alcohol dehydrogenase activity, or long-chain alcoholdehydrogenase activity), fatty alcohol oxidase activity, fatty aldehydedehydrogenase activity, and thioesterase activity.

Omega Oxidation—Monooxygenases

A cytochrome P450 enzyme (e.g., monooxygenase activity) often catalyzesthe insertion of one atom of oxygen into an organic substrate (RH) whilethe other oxygen atom is reduced to water. Insertion of the oxygen atomnear the omega carbon of a substrate yields an alcohol derivative of theoriginal starting substrate (e.g., yields a fatty alcohol). A cytochromeP450 sometimes is encoded by the host organism and sometimes can beadded to generate an engineered organism.

In certain embodiments, the monooxygenase activity is unchanged in ahost or engineered organism. In some embodiments, the host monooxygenaseactivity can be increased by increasing the number of copies of acytochrome P450 gene, or by increasing the activity of a promoter thatregulates transcription of a cytochrome P450 gene, thereby increasingthe production of target product (e.g., sebacic or dodecanedioic acid)due to increased carbon flux through the pathway. In certainembodiments, the cytochrome P450 gene can be isolated from any suitableorganism. Non-limiting examples of organisms that include, or can beused as donors for, cytochrome P450 enzymes include yeast (e.g.,Candida, Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).

The rate limiting step of ω-oxidation is the hydroxylation of theω-carbon of a fatty acid which is carried out by an enzyme systemcomposed of two enzymes, NADPH cytochrome P450 reductase (CPR) andcytochrome P450 monooxygenase (e.g., CYP52, EC 1.14.14.1). The P450'sare a gene family that produces isozymes with different substratespecificities. In Candida the gene family is typically composed ofCYP52A12, CYP52A13, CYP52A14, CYP52A15, CYP52A16, CYP52A17, CYP52A18,CYP52A19, CYP52A20, and CYP52D2. The P450 enzyme is encoded by a genefamily of CYP genes designated A12-A20, and D2 in Candida spp. Eachmember of the P450 gene family displays unique substrate chain-lengthspecificity. Using engineered Candida strains we have identified theP450 isozymes that improve performance upon different chain-length fattyacid feedstocks. For short- or medium-chain fatty acid feedstocks(C6-C14) CYP52A19 amplification improved performance more than the otherisozymes. For long-chain fatty acid feedstocks (>C16) CYP52A14amplification improved performance more than the other isozymes. In someembodiments, to increase the carbon flux through the w-oxidation pathwaythe enzyme activity for one or both of the CPR and the P450 enzymefamilies is amplified. In some embodiments, care is taken to select theP450 family member with substrate specificity that matches the chainlength of the exogenously supplied fatty acid feedstock. In someembodiments, to increase the carbon flux through the w-oxidation pathwaythe enzyme activity of a CYP52A19 is amplified. In some embodiments, toincrease the carbon flux through the w-oxidation pathway the enzymeactivity of a CYP52A14 is amplified.

The term “monooxygenase activity” as used herein refers to inserting oneatom of oxygen from O₂ into an organic substrate (RH) and reducing theother oxygen atom to water. In some embodiments, monooxygenase activityrefers to incorporation of an oxygen atom onto a six-carbon organicsubstrate. In certain embodiments, monooxygenase activity refers toconversion of hexanoate to 6-hydroxyhexanoic acid. Monooxygenaseactivity can be provided by any suitable polypeptide, such as acytochrome P450 polypeptide (hereafter “CYP450”) in certain embodiments.Nucleic acid sequences conferring CYP450 activity can be obtained from anumber of sources, including Bacillus megaterium and may be induced inorganisms including but not limited to Candida tropicalis, Yarrowialipolytica, Aspergillus nidulans, and Aspergillus parasiticus. Examplesof oligonucleotide sequences utilized to isolate a polynucleotidesequence encoding a polypeptide having CYP450 activity (e.g., CYP52A12polynucleotide, a CYP52A13 polynucleotide, a CYP52A14 polynucleotide, aCYP52A15 polynucleotide, a CYP52A16 polynucleotide, a CYP52A17polynucleotide, a CYP52A18 polynucleotide, a CYP52A19 polynucleotide, aCYP52A20 polynucleotide, a CYP52D2 polynucleotide, and/or a BM3polynucleotide) are presented herein. In some embodiments, monooxygenaseactivity is not altered in a host microorganism, and in certainembodiments, the activity is added or increased in the engineeredmicroorganism relative to the host microorganism. In some embodiments,the altered monooxygenase activity is an endogenous activity, and incertain embodiments, the altered monooxygenase activity is an exogenousactivity. In some embodiments, the exogenous activity is a singlepolypeptide with both monooxygenase and monooxygenase reductaseactivities (e.g., B. megaterium cytochrome P450:NADPH P450 reductase).

Presence, absence or amount of cytochrome P450 activity can be detectedby any suitable method known in the art. For example, detection can beperformed by assaying a reaction containing cytochrome P450 (CYP52Afamily) and NADPH-cytochrome P450 reductase (see Appl Environ Microbiol69: 5983 and 5992). Briefly, cells are grown under standard conditionsand harvested for production of microsomes, which are used to detect CYPactivity. Microsomes are prepared by lysing cells in Tris-bufferedsucrose (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.25M sucrose). Differentialcentrifugation is performed first at 25,000×g then at 100,000×g topellet cell debris then microsomes, respectively. The microsome pelletis resuspended in 0.1M phosphate buffer (pH 7.5), 1 mM EDTA to a finalconcentration of approximately 10 mg protein/mL. A reaction mixturecontaining approximately 0.3 mg microsomes, 0.1 mM sodium hexanoate, 0.7mM NADPH, 50 mM Tris-HCl pH 7.5 in 1 mL is initiated by the addition ofNADPH and incubated at 37° C. for 10 minutes. The reaction is terminatedby addition of 0.25 mL 5M HCl and 0.25 mL 2.5 ug/mL 10-hydroxydecanoicacid is added as an internal standard (3.3 nmol). The mixture isextracted with 4.5 mL diethyl ether under NaCl-saturated conditions. Theorganic phase is transferred to a new tube and evaporated to dryness.The residue is dissolved in acetonitrile containing 10 mM3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB) and 0.1 mL of 15mg/mL 18-crown-6 in acetonitrile saturated with K₂CO₃. The solution isincubated at 40° C. for 30 minutes before addition of 0.05 mL 2% aceticacid. The fluorescently labeled omega-hydroxy fatty acids are resolvedvia HPLC with detection at 430 nm and excitation at 355 nm (Yamada etal., 1991, Anal Biochem 199: 132-136). Optionally, specifically inducedCYP gene(s) may be detected by Northern blotting and/or quantitativeRT-PCR. (Craft et al., 2003, AppEnvironMicro 69: 5983-5991).

Omega Oxidation—Monooxygenase Reductases

A cytochrome P450 reductase (e.g., monooxygenase reductase activity)catalyzes the reduction of the heme-thiolate moiety in cytochrome P450by transferring an electron to the cytochrome P450. A cytochrome P450reductase sometimes is encoded by the host organism and sometimes can beadded to generate an engineered organism. In certain embodiments, themonooxygenase reductase activity is unchanged in a host or engineeredorganism. In some embodiments, the host monooxygenase reductase activitycan be increased by increasing the number of copies of a cytochrome P450reductase gene, or by increasing the activity of a promoter thatregulates transcription of a cytochrome P450 reductase gene, therebyincreasing the production of target product (e.g., sebacic ordodecanedioic acid) due to increased carbon flux through the pathway. Incertain embodiments, the cytochrome P450 reductase gene can be isolatedfrom any suitable organism. Non-limiting examples of organisms thatinclude, or can be used as donors for, cytochrome P450 reductase enzymesinclude yeast (e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,Nictotania, Cuphea).

The reductase (CPR) enzyme (EC 1.6.2.4) is able to work with any of theP450 isozymes. The reductase is encoded by the genes CPRA and CPRB inCandida sp. In some embodiments, to increase the carbon flux through theω-oxidation pathway the enzyme activity of a CPR is amplified. In someembodiments a CPRA gene is amplified. In some embodiments a CPRB gene isamplified.

The term “monooxygenase reductase activity” as used herein refers to thetransfer of an electron from NAD(P)H, FMN, or FAD by way of an electrontransfer chain, reducing the ferric heme iron of cytochrome P450 to theferrous state. The term “monooxygenase reductase activity” as usedherein also can refer to the transfer of a second electron via theelectron transport system, reducing a dioxygen adduct to a negativelycharged peroxo group. In some embodiments, a monooxygenase activity candonate electrons from the two-electron donor NAD(P)H to the heme ofcytochrome P450 (e.g., monooxygenase activity) in a coupled two-stepreaction in which NAD(P)H can bind to the NAD(P)H-binding domain of thepolypeptide having the monooxygenase reductase activity and electronsare shuttled from NAD(P)H through FAD and FMN to the heme of themonooxygenase activity, thereby regenerating an active monooxygenaseactivity (e.g., cytochrome P450). Monooxygenase reductase activity canbe provided by any suitable polypeptide, such as a cytochrome P450reductase polypeptide (hereafter “CPR”) in certain embodiments. Nucleicacid sequences conferring CPR activity can be obtained from and/orinduced in a number of sources, including but not limited to Bacillusmegaterium, Candida tropicalis, Yarrowia lipolytica, Aspergillusnidulans, and Aspergillus parasiticus. Examples of oligonucleotidesequences utilized to isolate a polynucleotide sequence encoding apolypeptide having CPR activity are presented herein. In someembodiments, monooxygenase reductase activity is not altered in a hostmicroorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism. In some embodiments, the altered monooxygenase reductaseactivity is an endogenous activity, and in certain embodiments, thealtered monooxygenase reductase activity is an exogenous activity. Insome embodiments, the exogenous activity is a single polypeptide withboth monooxygenase and monooxygenase reductase activities (e.g., B.megaterium cytochrome P450:NADPH P450 reductase).

Presence, absence or amount of CPR activity can be detected by anysuitable method known in the art. For example, an engineeredmicroorganism having an increased number of genes encoding a CPRactivity, relative to the host microorganism, could be detected usingquantitative nucleic acid detection methods (e.g., southern blotting,PCR, primer extension, the like and combinations thereof). An engineeredmicroorganism having increased expression of genes encoding a CPRactivity, relative to the host microorganism, could be detected usingquantitative expression based analysis (e.g., RT-PCR, western blotanalysis, northern blot analysis, the like and combinations thereof).Alternately, an enzymatic assay can be used to detect Cytochrome P450reductase activity, where the enzyme activity alters the opticalabsorbance at 550 nanometers of a substrate solution (Masters, B. S. S.,Williams, C. H., Kamin, H. (1967) Methods in Enzymology, X, 565-573).

Omega Oxidation—Hydroxy Fatty Acids

Omega-hydroxy fatty acids (HFAs) are intermediates in oxidation of theterminal methyl group of fatty acids (FIG. 31). HFAs can be producedduring the first oxidation step in the omega-oxidation pathway, which iscatalyzed by cytochrome P450 using molecular oxygen and electronssupplied by NADPH. Electron transfer from NADPH can be performed usingthe enzyme, cytochrome P450 reductase (CPR). HFAs can be furtheroxidized to form the omega-oxo-fatty acid. This oxidation of HFAs canoccur through three different enzymatic mechanisms: 1) Over-oxidation bycytochrome P450 which requires molecular oxygen, NADPH, and CPR; 2)Alcohol dehydrogenase (ADH), which requires either NAD+ or NADP+,depending upon the specificity of the ADH; or 3) Fatty alcohol oxidase(FAO), which requires molecular oxygen and produces hydrogen peroxide asa byproduct in the reaction. FAO enzymes are membrane-bound andassociated with peroxisomes in Candida. Omega-oxo-fatty acids can beoxidized to the dicarboxylic acid either through the over-oxidationreaction by cytochrome P450s or through the enzyme aldehydedehydrogenase (ALD).

HFAs are frequently found in small, but economically significant amountsin dicarboxylic acid fermentations in which beta-oxidation-blockedstrains of Candida using fatty acids or fatty acid methyl esters asfeedstock. Although HFAs only constitute approximately 5-10% of thefinal oxidation product, the presence of HFAs can result in decreasedyields and purity of a final fatty dicarboxylic acid product and can beundesirable.

Omega Oxidation—Alcohol Dehydrogenases

An alcohol dehydrogenase (e.g., fatty alcohol dehydrogenase, long-chainalcohol dehydrogenase) catalyzes the removal of a hydrogen from analcohol to yield an aldehyde or ketone and a hydrogen atom and NADH, inthe endoplasmic reticulum of a cell. In the case of longer chainalcohols (e.g., hexadecanol), water is utilized in the dehydrogenationto yield a long chain carboxylate, 2 NADH and H₂. An alcoholdehydrogenase sometimes is encoded by the host organism and sometimescan be added to generate an engineered organism. In certain embodiments,the alcohol dehydrogenase activity is unchanged in a host or engineeredorganism. In some embodiments, the host alcohol dehydrogenase activitycan be increased by increasing the number of copies of an alcoholdehydrogenase gene, or by increasing the activity of a promoter thatregulates transcription of an alcohol dehydrogenase gene, therebyincreasing the production of target product (e.g., sebacic ordodecanedioic acid) due to increased carbon flux through the pathway. Incertain embodiments, the alcohol dehydrogenase gene can be isolated fromany suitable organism. Non-limiting examples of organisms that include,or can be used as donors for, alcohol dehydrogenase enzymes includeyeast (e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,Nictotania, Cuphea). Non-limiting examples of fatty alcoholdehydrogenases are ADH1, ADH2a, ADH2b, ADH3, ADH4, ADH6, ADH7, ADH8,SFA1, FAO1, EC 1.1.1.66, EC 1.1.1.164 and/or EC 1.1.1.192. In someembodiments, the expression of ADH1, ADH2a, ADH2b, ADH3, ADH4, ADH6,ADH7, ADH8, SFA1, FAO1, EC 1.1.1.66, EC 1.1.1.164 and/or EC 1.1.1.192 isincreased in a fatty dicarboxylic acid producing organism.

Omega Oxidation—Fatty Alcohol Oxidases

A fatty alcohol oxidase (e.g., long-chain alcohol oxidase, EC 1.1.3.20)enzyme catalyzes the addition of oxygen to two molecules of a long-chainalcohol to yield 2 long chain aldehydes and 2 molecules of water, in theperoxisome of a cell. A fatty alcohol oxidase sometimes is encoded bythe host organism and sometimes can be added to generate an engineeredorganism. In certain embodiments, the fatty alcohol oxidase activity isunchanged in a host or engineered organism. In some embodiments, thehost fatty alcohol oxidase activity can be increased by increasing thenumber of copies of a fatty alcohol oxidase gene, or by increasing theactivity of a promoter that regulates transcription of a fatty alcoholoxidase gene, thereby increasing the production of target product (e.g.,sebacic or dodecanedioic acid) due to increased carbon flux through thepathway. In certain embodiments, the fatty alcohol oxidase gene can beisolated from any suitable organism. Non-limiting examples of fattyalcohol oxidases include FAO1, FAO2a, FAO2b, FAO13, FAO17, FAO18, FAO20and FAO1ΔPTS1. Non-limiting examples of organisms that include, or canbe used as donors for, fatty alcohol oxidase enzymes include yeast(e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma, Lodderomyces,Scheffersomyces, Clavispora, Yarrowia, Pichia, Kluyveromyces,Eremothecium, Zygosaccharomyces, Lachancea, Nakaseomyces), animals(e.g., Homo, Rattus), bacteria (e.g., Escherichia, Pseudomonas,Bacillus), or plants (e.g., Arabidopsis, Nictotania, Cuphea).

Omega Oxidation—Aldehyde Dehydrogenases

A fatty aldehyde dehydrogenase (e.g., long chain aldehyde dehydrogenase)enzyme catalyzes the oxidation of long chain aldehydes to a long chaindicarboxylic acid, NADH and H₂. A fatty aldehyde dehydrogenase sometimesis encoded by the host organism and sometimes can be added to generatean engineered organism. In certain embodiments, the fatty aldehydedehydrogenase activity is unchanged in a host or engineered organism. Insome embodiments, the host fatty aldehyde dehydrogenase activity can beincreased by increasing the number of copies of a fatty aldehydedehydrogenase gene, or by increasing the activity of a promoter thatregulates transcription of a fatty aldehyde dehydrogenase gene, therebyincreasing the production of target product (e.g., sebacic ordodecanedioic acid) due to increased carbon flux through the pathway. Incertain embodiments, the fatty aldehyde dehydrogenase gene can beisolated from any suitable organism. Non-limiting examples of organismsthat include, or can be used as donors for, fatty aldehyde dehydrogenaseenzymes include yeast (e.g., Candida, Saccharomyces, Debaryomyces,Meyerozyma, Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,Nictotania, Cuphea). Non-limiting examples of aldehyde dehydrogenasesare ALD1, ALD5, HFD1, HFD1a, EC 1.2.1.3, EC 1.2.1.48 and/or HFD2. Insome embodiments, the expression of ALD1, ALD5, HFD1 and/or HFD2 isincreased in a fatty dicarboxylic acid producing organism.

Omega Oxidation—Thioesterases

A thioesterase enzyme (e.g., acyl-CoA thioesterase activity, acyl-ACPthioesterase activity) catalyzes the removal of Coenzyme A or acylcarrier protein (e.g., ACP) from a fatty acid including acyl-CoA or acylcarrier protein (e.g., esterified fatty acid) to yield a fatty acid andan alcohol. The reaction occurs in the presence of water and Coenzyme Aor acyl carrier protein is specifically removed at a thiol group. Athioesterase sometimes is encoded by the host organism and sometimes canbe added to generate an engineered organism. In certain embodiments, thethioesterase activity is unchanged in a host or engineered organism. Insome embodiments, the host thioesterase activity can be increased byincreasing the number of copies of a thioesterase gene, or by increasingthe activity of a promoter that regulates transcription of athioesterase gene, thereby increasing the production of target product(e.g., sebacic or dodecanedioic acid) due to increased carbon fluxthrough the pathway. In certain embodiments, a thioesterase gene can beisolated from any suitable organism. Non-limiting examples of organismsthat include, or can be used as donors for, thioesterase enzymes includeyeast (e.g., Candida, Saccharomyces, Debaryomyces, Meyerozyma,Lodderomyces, Scheffersomyces, Clavispora, Yarrowia, Pichia,Kluyveromyces, Eremothecium, Zygosaccharomyces, Lachancea,Nakaseomyces), animals (e.g., Homo, Rattus), bacteria (e.g.,Escherichia, Pseudomonas, Bacillus), or plants (e.g., Arabidopsis,Nictotania, Cuphea).

Engineered Pathways

FIGS. 1-8 depict embodiments of biological pathways for making sebacicacid and dodecanedioic acid, using various alkanes, fatty acids, fattyalcohols or combinations thereof. Any suitable alkane, fatty acid, fattyalcohol, plant based oil, seed based oil, non-petroleum derived soapstock or the like can be used as the feedstock for the organism (e.g.,dodecane, methyl laurate, lauric acid, carbon sources having 10 orgreater carbons (e.g. for sebacic acid production) or carbon sourceshaving 12 or greater carbons (e.g. for dodecanedioic acid production).In some embodiments, carbon sources with greater than 12 carbons can bemetabolized using naturally occurring and/or engineered pathways toyield molecules that can be further metabolized using the beta oxidationpathway shown in the lower portion of FIGS. 5-8. In some embodiments,the activities in the pathways depicted in FIGS. 1-8 can be engineered,as described herein, to enhance metabolism and target product formation.

In certain embodiments, one or more activities in one or more metabolicpathways can be engineered to increase carbon flux through theengineered pathways to produce a desired product (e.g., sebacic ordodecanedioic acid). The engineered activities can be chosen to allowincreased production of metabolic intermediates that can be utilized inone or more other engineered pathways to achieve increased production ofa desired product with respect to the unmodified host organism. Theengineered activities also can be chosen to allow decreased activity ofenzymes that reduce production of a desired intermediate or end product(e.g., reverse activities). This “carbon flux management” can beoptimized for any chosen feedstock, by engineering the appropriateactivities in the appropriate pathways. Non-limiting examples are givenherein using pure alkanes (e.g., single chain length alkanes, dodecaneor example), mixed chain-length alkanes, long-chain alkanes, pure fattyacids (e.g., single chain length fatty acids, capric acid for example)and mixed chain length fatty acids (see FIGS. 1-8). The process of“carbon flux management” through engineered pathways produces adicarboxylic acid (e.g. sebacic acid or dodecanedioic acid) at a leveland rate closer to the calculated maximum theoretical yield for anygiven feedstock, in certain embodiments. The terms “theoretical yield”or “maximum theoretical yield” as used herein refer to the yield ofproduct of a chemical or biological reaction that can be formed if thereaction went to completion. Theoretical yield is based on thestoichiometry of the reaction and ideal conditions in which startingmaterial is completely consumed, undesired side reactions do not occur,the reverse reaction does not occur, and there no losses in the work-upprocedure.

A microorganism may be modified and engineered to include or regulateone or more activities in a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) pathway.The term “activity” as used herein refers to the functioning of amicroorganism's natural or engineered biological pathways to yieldvarious products including a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) and itsprecursors. A fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid)producing activity can be provided by any non-mammalian source incertain embodiments. Such sources include, without limitation,eukaryotes such as yeast and fungi and prokaryotes such as bacteria. Insome embodiments, a reverse activity in a pathway described herein canbe altered (e.g., disrupted, reduced) to increase carbon flux through abeta oxidation pathway, an omega oxidation pathway, or a beta oxidationand omega oxidation pathway, towards the production of target product(e.g., sebacic or dodecanedioic acid). In some embodiments, a geneticmodification disrupts an activity in the beta oxidation pathway, ordisrupts a polynucleotide that encodes a polypeptide that carries out aforward reaction in the beta oxidation pathway, which renders betaoxidation activity undetectable. The term “undetectable” as used hereinrefers to an amount of an analyte that is below the limits of detection,using detection methods or assays known (e.g., described herein). Incertain embodiments, the genetic modification partially reduces betaoxidation activity. The term “partially reduces beta oxidation activity”as used here refers to a level of activity in an engineered organismthat is lower than the level of activity found in the host or startingorganism.

In some embodiments, a beta-oxidation activity can be modified to alterthe catalytic specificity of the chosen activity. In certainembodiments, an acyl-CoA oxidase activity can be altered by modifying acatalytic domain associated with carbon chain length preference and/orspecificity. In some embodiments, the altered catalytic specificity canbe found by screening naturally occurring variant or mutant populationsof a host organism. In certain embodiments, the altered catalytic can begenerated by various mutagenesis techniques in conjunction withselection and/or screening for the desired activity. In someembodiments, the altered catalytic activity can be generated bygenerating chimeric acyl-CoA oxidases using a mix and match approach,followed by selection and/or screening for the desired catalyticactivity. Examples of experiments performed to generate acyl-CoAoxidases with altered catalytic activity are described herein.

An activity within an engineered microorganism provided herein caninclude one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or all) of the following activities: 6-oxohexanoic acid dehydrogenaseactivity; 6-hydroxyhexanoic acid dehydrogenase activity; cytochrome P450activity; cytochrome P450 reductase activity; fatty alcohol oxidaseactivity; acyl-CoA ligase activity, acyl-CoA oxidase activity; enoyl-CoAhydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, fatty acidsynthase activity, lipase activity, acetyl-CoA carboxylase activity,acyltransferase activity (diacylglycerol acyl transferase,lecithin-cholesterol acyltransferase, phospholipid:diacylglycerolacyltransferase) and thioesterase activity (e.g., acyl-CoA hydrolase,acyl-CoA thioesterase, acyl-ACP thioesterase, acetyl-CoAC-acyltransferase, beta-ketothiolase, and the like). In certainembodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all) ofthe foregoing activities is altered by way of a genetic modification. Insome embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14 or all) of the foregoing activities is altered by way of (i)adding a heterologous polynucleotide that encodes a polypeptide havingthe activity, and/or (ii) altering or adding a regulatory sequence thatregulates the expression of a polypeptide having the activity. Incertain embodiments, one or more of the foregoing activities is alteredby way of (i) disrupting an endogenous polynucleotide that encodes apolypeptide having the activity (e.g., insertional mutagenesis), (ii)deleting a regulatory sequence that regulates the expression of apolypeptide having the activity, and/or (iii) deleting the codingsequence that encodes a polypeptide having the activity (e.g., knock outmutagenesis).

The term “omega hydroxyl fatty acid dehydrogenase activity” as usedherein refers to conversion of an omega hydroxyl fatty acid to an omegaoxo fatty acid. The omega hydroxyl fatty acid dehydrogenase activity canbe provided by a polypeptide. In some embodiments, the polypeptide isencoded by a heterologous nucleotide sequence introduced to a hostmicroorganism. In certain embodiments, an endogenous polypeptide havingthe omega hydroxyl fatty acid dehydrogenase activity is identified inthe host microorganism, and the host microorganism is geneticallyaltered to increase the amount of the polypeptide produced (e.g., aheterologous promoter is introduced in operable linkage with apolynucleotide that encodes the polypeptide; the copy number of apolynucleotide that encodes the polypeptide is increased (e.g., byintroducing a plasmid that includes the polynucleotide)). Nucleic acidsequences conferring omega hydroxyl fatty acid dehydrogenase activitycan be obtained from a number of sources, including Actinobacter,Norcardia, Pseudomonas and Xanthobacter bacteria. Examples of an aminoacid sequence of a polypeptide having omega hydroxyl fatty aciddehydrogenase activity and a nucleotide sequence of a polynucleotidethat encodes the polypeptide, are presented herein. Presence, absence oramount of omega hydroxyl fatty acid dehydrogenase activity can bedetected by any suitable method known in the art. In some embodiments,omega hydroxyl fatty acid dehydrogenase activity is not altered in ahost microorganism, and in certain embodiments, the activity is added orincreased in the engineered microorganism relative to the hostmicroorganism.

Increasing NADPH Production in Yeast Producing a Fatty Dicarboxylic Acid

The ω-oxidation pathway requires the cofactors NADPH in the first stepand NAD⁺ in the second and third steps. Since the first step inω-oxidation is the rate-limiting step, amplification of the enzymeactivity performing this step in the cell would also require asufficient supply of the NADPH cofactor for the reaction. There are anumber of cellular reactions that produce NADPH that may be used by thefirst step in ω-oxidation. Some of the enzymes performingNADPH-producing reactions in the cell are glucose-6-phosphatedehydrogenase, isocitrate dehydrogenase, and glycerol-3-phosphatedehydrogenase. Amplification of the activity levels of any of thesegenes can increase cellular levels of NADPH to provide enough cofactorfor an amplified w-oxidation activity.

NADPH is required for both ω-oxidation and fatty acid synthesis. Geneticchanges that increase the amount of NADPH in the cell can result in aproduction boost for the production of diacid from either single fattyacids and/or fatty acids mixtures. In addition, if the number of NADPHobtained per glucose is increased, the amount of glucose required asco-feed can be reduced.

Increasing NADPH Production by Increasing Glucose-6-PhosphateDehydrogenase Activity Through Overexpression of ZWF1 or ZWF2 Genes

The ZWF1 and ZWF2 genes encode two isozymes of glucose-6-phosphatedehydrogenase (G6PDH, e.g., EC 1.1.1.49). In S. cerevisiae increasingglucose-6-phosphate dehydrogenase (G6PDH) activity results in anincrease in cytosolic NADPH. This technique has been used to createstrains with increased xylitol production and increased furfuralsresistance. In some embodiments the ZWF1 open reading frame will beamplified from either Candida strain ATCC20336 or Scheffersomycesstipitis and placed under the control of the ZWF1 promoter, TEF1promoter, POX4 promoter, or another strong constitutive or induciblepromoter. These cassettes can be transformed into suitable yeast strainsfor either specific or random integration using the URA3 auxotrophicmarker for selection. Ura+ strains can be analyzed by PCR and qPCR forproper integration or copy number. Increased glucose-6-phosphatedehydrogenase activity can be confirmed by activity assays. Strains canthen be tested for production of the desired fatty dicarboxylic acid. Insome embodiments strains can then be tested for production of aoctanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid,suberic or adipic acid depending on the strain and feedstock used. Thefermentation performance of a yeast strain engineered for increasedNADPH production can be compared to the parental strain.

Two examples of the amino acid sequences for G6PDH are shown below:

-   >Scheffersomyces_stipitis_ZWF1—SEQ ID NO: 157-   Candida strain ATCC20336_ZWF1—SEQ ID NO: 74

Increasing NADPH Production by Decreasing Glycolysis and IncreasingPentose Pathway Through Disruption of the PGI1 Gene.

In some embodiments a disruption cassette for PGI1 is constructed. Forexample 300 to 700 bp of the 5′ and 3′ untranslated region or openreading frame of the PGI1 gene can be amplified. The two pieces can beligated together leaving a unique restriction site between them where anURA3 can be cloned into. This URA3 cassette can have either theterminator or promoter duplicated in either the beginning or the end ofthe URA3 cassette, respectively. The direct repeat can allow loop-out ofthe URA3. The disruption cassette can then be transformed into asuitable yeast strain and select by growing in uracil deficient plates.Disruption of the first copy of PGI1 can be verified by PCR. URA3loopout events can be selected by growth in 5-Fluorootic acid containingplates. The loop-out event can be verified by PCR using primers outsidethe region encompassing the transformation cassette. This strain can betransformed with the PGI1 disruption cassette previously used or a newdisruption cassette that targets regions not present in the firstdisruption. Ura+ strains can be screened for the complete loss of thePGI1 gene. Strains can then be tested for production of the desiredfatty dicarboxylic acid. In some embodiments strains can then be testedfor production of an octanedioic acid, decanedioic acid, dodecanedioicacid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid, suberic or adipic acid depending on the strain andfeedstock used. The fermentation performance of a yeast strainengineered for increased NADPH production can be compared to theparental strain.

An example of the amino acid sequences for PGI1 is shown below:

-   Candida strain ATCC20336_PGI1—SEQ ID NO: 78

Increasing NADPH Production by Overexpression of KIGDP1

GDP1 (e.g., GDP1 of Kluyveromyces lactis, i.e., KIGDP1) encodes an NADP+depending glyceraldehyde dehydrogenase (EC 1.2.1.9) that convertsglyceraldehyde 3-phosphate into 1,3 biphosphoglycerate producing NADPHinstead of NADH. This activity can increase the production of NADPH fromglucose. KIGDP1 open reading can be mutagenized to change the CTG codonto another leucine encoding codon. The open reading frame can be placedunder the control of the TEF1 promoter, POX4 promoter or another strongconstitutive or inducible promoter. These cassettes can be integratedinto any suitable yeast strain by targeted or random integration usingthe URA3 auxotrophic marker to select for transformation events. Ura+strains can be analyzed by PCR and qPCR for proper integration or copynumber determination. In addition, increased NADP+ dependentglyceraldehyde 3-phosphate dehydrogenase activity can be confirmed byactivity assay. Strains can then be tested for production of the desiredfatty dicarboxylic acid. In some embodiments strains can then be testedfor production of an octanedioic acid, decanedioic acid, dodecanedioicacid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid, suberic or adipic acid depending on the strain andfeedstock used. The fermentation performance of a yeast strainengineered for increased NADPH production can be compared to theparental strain.

An example of the amino acid sequence for GDP1 is shown below:

-   >GDP1, KI—SEQ ID NO: 72

Increasing NADPH Production by Overexpression of IDPs

IDP1 and IDP2 (e.g., from Candida strain 20336) encode proteins with anisocitrate dehydrogenase activity that converts isocitrate toα-ketoglutarate producing NADPH instead of NADH (e.g., EC 1.1.1.42). TheIDP1 protein is targeted to the mitochondria while the IDP2 protein istargeted to the peroxisome and it can be present in the ER wherew-oxidation happens. IDP2 expression has been shown to be induced by thepresence of alkanes and overexpression may increase NADPH availability.

The open reading frame can be placed under the TEF1 promoter, POX4promoter, or another strong constitutive or inducible promoter. Thesecassettes can be integrated into any suitable yeast strains either bytargeted integration or random integration using the URA3 auxotrophicmarker to select for transformation events. Ura+ strains can be verifiedby PCR and qPCR for proper integration or copy number. In addition,increased NADP+ dependent isocitrate dehydrogenase activity can beconfirmed by activity assay. Strains can then be tested for productionof the desired fatty dicarboxylic acid. In some embodiments strains canthen be tested for production of an octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid, suberic or adipic aciddepending on the strain and feedstock used. The fermentation performanceof a yeast strain engineered for increased NADPH production can becompared to the parental strain.

Another IDP to be tested can be IDP3 (e.g. from Saccharomycescerevisiae, i.e., Scldp3). This protein is targeted to the peroxisomeand may also be present in the ER. A similar approach can be taken forIDP2 except that the open reading frame may need to be mutagenized ifthere are any CTG codons.

An example of the amino acid sequences for an IDP2 and IDP3 are shownbelow:

-   Candida strain ATCC20336 IDP2—SEQ ID NO: 67-   >Saccharomyces_cerevisiae_IDP3—SEQ ID NO: 69

Increasing NADPH Production by Overexpression of ScMAE1 and ScPYC2

MAE encodes a malic enzyme (e.g., 1.1.1.40) converting malic acid topyruvate producing NADPH (as shown below).

(S)-malate+NADP+→pyruvate+CO2+NADPH+H+

When overexpressed in the cytosol in the presence of PYC2 (i.e.,pyruvate carboxylase, e.g., 6.4.1.1) that converts pyruvate tooxaloacetate) a shunt is formed that produces one NADPH at the expenseof one ATP and NADH. MAE expression can be directed to the cytosol byexpressing a truncated version that prevents its translocation into themitochondria.

MAE1 (e.g., from a Candida strain or Saccharomyces cerevisiae, i.e.,ScMAE1) and PYC2 (e.g., from Saccharomyces cerevisiae, i.e., ScPYC2)open reading frames can be amplified and mutagenized to replace any CTGcodons for other leucine encoding codons. The genes can be placed underthe control of the TEF1 promoter, POX4 promoter, or another strongconstitutive or inducible promoter. These cassettes can be integratedinto any suitable yeast strain by targeting integration or randomintegration using the URA3 auxotrophic marker to select fortransformation events. Ura+ strains can be verified by PCR and qPCR forproper integration or copy number. Strains can then be tested forproduction of the desired fatty dicarboxylic acid. In some embodimentsstrains can then be tested for production of an octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid, subericor adipic acid depending on the strain and feedstock used. Thefermentation performance of a yeast strain engineered for increasedNADPH production can be compared to the parental strain.

An example of the amino acid sequences for a ScMAE1 and ScPYC2 are shownbelow:

-   >ScMAE1—SEQ ID NO: 191-   >ScPYC2—SEQ ID NO: 107-   >Candida strain, truncated cytosolic MAE1—SEQ: ID 143

Increasing NADPH Production when Using Glycerol as a Co-Feed

Archaeoglobus fulgidus gpsA encodes a glycerol 3-phosphate dehydrogenaseusing NADP+ as a co-factor. The gene encoding this enzyme can bemutagenized to change any CTG codons to other leucine encoding codons.This gene can be placed under either a constitutive or or glycerolinducible promoter with a loop-out capable URA3 auxotrophic marker in adisruption cassette for GUT2. This cassette can be transformed into anysuitable yeast strain disrupting the first copy of GUT2. The URA3 markercan be recycled and the resulting strain can be retransformed with theintegration cassette. Strains that have both copies of GUT2 disruptedcan be selected. This strain should produce NADPH instead of FADH in theconversion of glycerol-3-phosphate to dihydroxyacetone. Strains can thenbe tested for production of the desired fatty dicarboxylic acid. In someembodiments strains can then be tested for production of an octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid, subericor adipic acid depending on the strain and feedstock used. Thefermentation performance of a yeast strain engineered for increasedNADPH production can be compared to the parental strain.

An example of the amino acid sequences for a GUT2 and Archaeoglobusfulgidus gpsA are shown below:

-   >Candida strain ATCC20336 GUT2—SEQ ID NO: 109-   >AfgpsA—SEQ ID NO: 111

Acyl-CoA Oxidases

The term “acyl-CoA oxidase activity” as used herein refers to theoxidation of a long chain fatty-acyl-CoA to a trans-2,3-dehydroacyl-CoAfatty alcohol. In some embodiments, the acyl-CoA activity is from aperoxisome. In certain embodiments, the acyl-CoA oxidase activity is aperoxisomal acyl-CoA oxidase (POX) activity, carried out by a POXpolypeptide. In some embodiments the acyl-CoA oxidase activity isencoded by the host organism and sometimes can be altered to generate anengineered organism. Acyl-CoA oxidase activity is encoded by the POX4and POX5 genes of C. tropicalis. In certain embodiments, endogenousacyl-CoA oxidase activity can be increased. In some embodiments,acyl-CoA oxidase activity of the POX4 polypeptide or the POX5polypeptide can be altered independently of each other (e.g., increaseactivity of POX4 alone, POX5 alone, increase one and disrupt the other,and the like). Increasing the activity of one POX activity, whiledisrupting the activity of another POX activity, may alter the specificactivity of acyl-CoA oxidase with respect to carbon chain length, whilemaintaining or increasing overall flux through the beta oxidationpathway, in certain embodiments.

In certain embodiments, host acyl-CoA oxidase activity of one of the POXgenes can be increased by genetically altering (e.g., increasing) theamount of the polypeptide produced (e.g., a strongly transcribed orconstitutively expressed heterologous promoter is introduced in operablelinkage with a polynucleotide that encodes the polypeptide; the copynumber of a polynucleotide that encodes the polypeptide is increased(e.g., by introducing a plasmid that includes the polynucleotide,integration of additional copies in the host genome)). In someembodiments, the host acyl-CoA oxidase activity can be decreased bydisruption (e.g., knockout, insertion mutagenesis, the like andcombinations thereof) of an acyl-CoA oxidase gene, or by decreasing theactivity of the promoter (e.g., addition of repressor sequences to thepromoter or 5′UTR) which transcribes an acyl-CoA oxidase gene.

As noted above, disruption of nucleotide sequences encoding POX4, POX 5,or POX4 and POX5 sometimes can alter pathway efficiency, specificityand/or specific activity with respect to metabolism of carbon chains ofdifferent lengths (e.g., carbon chains including fatty alcohols, fattyacids, paraffins, dicarboxylic acids of between about 1 and about 60carbons in length). In some embodiments, the nucleotide sequence ofPOX4, POX5, or POX4 and POX5 is disrupted with a URA3 nucleotidesequence encoding a selectable marker, and introduced to a hostmicroorganism, thereby generating an engineered organism deficient inPOX4, POX5 or POX4 and POX5 activity. Nucleic acid sequences encodingPOX4 and POX5 can be obtained from a number of sources, includingCandida tropicalis, for example. Examples of POX4 and POX5 amino acidsequences and nucleotide sequences of polynucleotides that encode thepolypeptides, are presented herein. Described in the examples areexperiments conducted to amplify the activity encoded by the POX5 gene.

Also as noted above, catalytic specificity of acyl-CoA oxidases (e.g.,POX4, POX5) can be altered by a variety of methods. Altering the bindingand/or catalytic specificity of acyl-CoA oxidases may prove advantageousfor generating novel acyl-CoA oxidases with altered chain lengthrecognition, altered chain length catalytic activity, and/or generationof an acyl-CoA oxidase activity with a narrow or specific chain lengthspecificity, thereby allowing further increases in pathway efficiency,specificity and/or specific activity with respect to metabolism ofcarbon chains of different lengths or metabolism of carbon chaindistributions found in a particular chosen feedstock. In someembodiments the altered acyl-CoA oxidase sequences are identified and/orgenerated by; (i) screening naturally occurring variant populations;(ii) mutagenesis of endogenous sequences; (iii) introduction ofheterologous sequences having a desired specificity; (iv) generation ofchimeric sequences having a portion of the coding sequence from onepolynucleotide source (e.g., gene, organism) and a portion of the codingsequence from another source and/or (v) intelligent design usingnucleotide sequences and three dimensional structure analysis from anacyl-CoA oxidase having a desired specificity to remodel an endogenousacyl-CoA oxidase, thereby generating a novel specificity enzyme. In someembodiments a chimeric acyl-CoA oxidase sequence can have polynucleotidesequence contributions from two or more sources. In some embodiments, achimeric acyl-CoA oxidase sequence comprises a portion of the codingsequences from an endogenous polynucleotide and a portion of the codingsequence from a heterologous polynucleotide. Described in the examplesare methods utilized to identify and/or generate acyl-CoA oxidases withnovel catalytic and binding specificities.

Presence, absence or amount of POX4 and/or POX5 activity can be detectedby any suitable method known in the art. For example, using enzymaticassays as described in Shimizu et al, 1979, and as described herein inthe Examples. Alternatively, nucleic acid sequences representing nativeand/or disrupted POX4 and POX5 sequences also can be detected usingnucleic acid detection methods (e.g., PCR, primer extension, nucleicacid hybridization, the like and combinations thereof), or quantitativeexpression based analysis (e.g., RT-PCR, western blot analysis, northernblot analysis, the like and combinations thereof), where the engineeredorganism exhibits decreased RNA and/or polypeptide levels as compared tothe host organism.

Thioesterase

The term “thioesterase activity” as used herein refers to removal ofCoenzyme A from hexanoate. The term “thioesterase activity” as usedherein also refers to the removal of Coenzyme A from an activated fattyacid (e.g., fatty-acyl-CoA). A Non-limiting example of an enzyme withthioesterase activity includes acyl-CoA hydrolase (e.g., EC 3.1.2.20;also referred to as acyl coenzyme A thioesterase, acyl-CoA thioesterase,acyl coenzyme A hydrolase, thioesterase B, thioesterase II, lecithinaseB, lysophopholipase L1, acyl-CoA thioesterase 1, and acyl-CoAthioesterase). Thioesterases that remove Coenzyme A from fatty-acyl-CoAmolecules catalyze the reaction,

acyl-CoA+H₂O—→CoA+a carboxylate,

where the carboxylate often is a fatty acid. The released Coenzyme A canthen be reused for other cellular activities.

The thioesterase activity can be provided by a polypeptide. In certainembodiments, the polypeptide is an endogenous nucleotide sequence thatis increased in copy number, operably linked to a heterologous and/orendogenous promoter, or increased in copy number and operably linked toa heterologous and/or endogenous promoter. In some embodiments, thepolypeptide is encoded by a heterologous nucleotide sequence introducedto a host microorganism. Nucleic acid sequences conferring thioesteraseactivity can be obtained from a number of sources, including Cuphealanceolata, C. tropicalis (e.g., see SEQ ID NOS: 33 and 35), and E. coli(e.g., see SEQ ID NO: 37). Additional organisms that can be used asthioesterase polynucleotide sequence donors are given herein. Examplesof such polypeptides include, without limitation, acyl-(ACP)thioesterase type B from Cuphea lanceolata (see SEQ ID NO: 1), acyl-CoAhydrolase (e.g., ACHA and ACHB, see SEQ ID NOS: 34 and 36)) from C.tropicalis, acyl-CoA thioesterase (e.g., TESA, see SEQ ID NO: 38) fromE. coli. A non-limiting example of a thioesterase polynucleotidesequences is referenced by accession number CAB60830 at the World WideWeb Uniform Resource Locator (URL) ncbi.nlm.nih.gov of the NationalCenter for Biotechnology Information (NCBI).

Presence, absence or amount of thioesterase activity can be detected byany suitable method known in the art. An example of such a method isdescribed Chemistry and Biology 9: 981-988. In some embodiments,thioesterase activity is not altered in a host microorganism, and incertain embodiments, the activity is added or increased in theengineered microorganism relative to the host microorganism. In someembodiments, a polypeptide having thioesterase activity is linked toanother polypeptide (e.g., a hexanoate synthase A or hexanoate synthaseB polypeptide). Non-limiting examples of polynucleotide sequencesencoding thioesterase activities and polypeptides having thioesteraseactivity are provided in Example 33.

Reducing Omega Fatty Acid Conversion—General

The term “a genetic modification that reduces omega hydroxyl fatty acidconversion” as used herein refer to genetic alterations of a hostmicroorganism that reduce an endogenous activity that converts an omegahydroxyl fatty acid to another product. In some embodiments, anendogenous omega hydroxyl fatty acid dehydrogenase activity is reduced.Such alterations can advantageously increase the amount of adicarboxylic acid, which can be purified and further processed.

Reducing Beta Oxidation—General

The term “a genetic modification that reduces beta-oxidation activity”as used herein refers to a genetic alteration of a host microorganismthat reduces an endogenous activity that oxidizes a beta carbon ofcarboxylic acid containing organic molecules. In certain embodiments,the organic molecule is a ten or twelve carbon molecule, and sometimescontains one or two carboxylic acid moieties located at a terminus ofthe molecule (e.g., sebacic or dodecanedioic acid). Such alterations canadvantageously increase yields of end products, such as a fattydicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid).

Increasing Fatty Acid Synthesis—General

The term “a genetic modification that results in increased fatty acidsynthesis” as used herein also refers to a genetic alteration of a hostmicroorganism that reduces an endogenous activity that converts fattyacids into fatty-acyl-CoA intermediates. In some embodiments, anendogenous activity that converts fatty acids into fatty-acyl-CoAintermediates is reduced. In certain embodiments, an acyl-CoA synthetaseactivity is reduced. Such alterations can advantageously increase yieldsof end products, such as a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid).

Acyl-CoA Synthetase

Organisms that have a complete block of the β-oxidation pathway cannotutilize fatty acids or diacids for energy. In these β-oxidation blockedorganisms, the chain length of the diacid produced mimics the chainlength of the fatty acid feedstock. Blocking the β-oxidation pathwayremoves the primary route for diacid product yield loss. In someembodiments, genetic modifications that alter the cell's ability toutilize fatty acids in other biochemical pathways results in increaseddiacid production. In some embodiments, blocking a fatty acid activationpathway by knocking out or modifying an acyl CoA synthetase results inincreased diacid production.

The activation of fatty acids to fatty acyl-CoA thioesters is performedby an enzyme called acyl-CoA synthetase (ACS). Acyl-CoA synthetases area member of the ligase class of enzymes and catalyzes the reaction,

ATP+Fatty Acid+CoA<=>AMP+Pyrophosphate+Fatty-Acyl-CoA.

Fatty acids can be converted into fatty-acyl-CoA intermediates by theactivity of an acyl-CoA synthetase (e.g., ACS1, ACS2; EC 6.2.1.3; alsoreferred to as acyl-CoA synthetase, acyl-CoA ligase), in many organisms.Yeast cells contain multiple genes for ACS enzymes that are targeted todifferent cellular locations and may have different substratechain-length specificities. S. cerevisiae has six genes with ACSactivity named FAA1, FAA2. FAA3, FAA4, FAT1, and FAT2. The correspondingproteins produced by these genes are often called Faa1p, Faa2p, Faa3p,Faa4p, Fat1p and Fat2p respectively. The Faa1p isozyme can exhibit broadsubstrate chain-length specificity, represents 90% of the cellular ACSactivity, and is localized in the cytosolic and microsomal fractions.The Faa2p isozyme is targeted to the peroxisome and has broadchain-length specificity. The Faa3p isozyme has a substrate specificityfor long-chain or very long-chain fatty acids and its cellularlocalization is unknown. Faa4p has broad chain-length specificity andhas been shown to be important in protein myristoylation. Fat1p is adual function protein localized to the cellular membrane that hasactivity for both fatty acid transport and fatty acid activation. Fat2pis targeted to the peroxisomal membrane for medium chain fatty acidtransport and activation.

Acyl-CoA synthetase has six isoforms encoded by ACS1, FAT1, ACS2A,ACS2B, ACS2C, and ACS2D, respectively, in some Candida spp. (e.g.,homologous to FAA1, FAT1, and FAA2 in S. cerevisiae).

Disruption of the genes encoding ACS isozymes with activity targeted tothe cellular membrane and to the cytosolic fraction can leave theexogenously supplied fatty acids in the free fatty acid form which is asubstrate for entry into the w-oxidation pathway. This essentiallyredirects exogenously supplied fatty acids from normal cellularutilization (energy, triacylglycerides, phospholipids) to the productionof the desired diacid product. In some embodiments, in Candida strainATCC20336 these gene targets are ACS1 and FAT1.

-   -   Candida strain ATCC20336 ACS1—SEQ ID NO: 40    -   Candida strain ATCC20336 FAT1—SEQ ID NO: 148

Disruption of the genes encoding ACS isozymes with activity targeted tothe peroxisome can prevent the activation of any exogenously suppliedfatty acids that are transported to the interior of the peroxisomalcompartment. In a 8-oxidation blocked organism fatty acyl-CoA moleculescannot enter 3-oxidation, but they can be substrates for the synthesisof phospholipids. Knocking out the genes encoding these ACS isozymes canincrease the yield of a diacid product by redirecting the free fattyacids to w-oxidation instead of the phospholipid synthesis pathway.Candida strain ATCC20336 homologs to the peroxisomal S. cerevisiae FAA2are named ACS2A, ACS2B, ACS2C, and ACS2D and the protein sequenes ofACS2A, ACS2B and ACS2C are shown below.

-   -   Candida strain ATCC20336_ACS2A—SEQ ID NO: 80    -   Candida strain ATCC20336_ACS2B—SEQ ID NO: 158    -   Candida strain ATCC20336_ACS2C—SEQ ID NO: 159

Fatty acids and Coenzyme A often are utilized in the activation of fattyacids to fatty-acyl-CoA intermediates for entry into various cellularprocesses. Without being limited by theory, it is believed thatreduction in the amount of fatty-acyl-CoA available for various cellularprocesses can increase the amount of fatty acids available forconversion into a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) by otherengineered pathways in the same host organism (e.g., omega oxidationpathway, beta oxidation pathway, omega oxidation pathway and betaoxidation pathway). Acyl-CoA synthetase can be inactivated by anysuitable means. Described herein are gene knockout methods suitable foruse to disrupt the nucleotide sequence that encodes a polypeptide havingACS1 activity. A nucleotide sequence of ACS1 is provided in Example 33,SEQ ID NO: 39. An example of an integration/disruption construct,configured to generate a deletion mutant for ACS1 is also provided inthe Examples.

The presence, absence or amount of acyl-CoA synthetase activity can bedetected by any suitable method known in the art. Non-limiting examplesof suitable detection methods include enzymatic assays (e.g., Lageweg etal “A Fluorimetric Assay for Acyl-CoA Synthetase Activity”, AnalyticalBiochemistry, 197(2):384-388 (1991)), PCR based assays (e.g., qPCR,RT-PCR), immunological detection methods (e.g., antibodies specific foracyl-CoA synthetase), the like and combinations thereof.

The term “a genetic modification that results in increased fatty acidsynthesis” as used herein also refers to a genetic alteration of a hostmicroorganism that reduces an endogenous activity that converts longchain and very long chain fatty acids into activated fatty-acyl-CoAintermediates. In some embodiments, an endogenous activity that convertslong chain and very long chain fatty acids into activated fatty-acyl-CoAintermediates is reduced. In certain embodiments, a long chain acyl-CoAsynthetase activity is reduced. Such alterations can advantageouslyincrease yields of end products, such as a fatty dicarboxylic acid(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid).

Long chain fatty acids (e.g., C12-C18 chain lengths) and very long chainfatty acids (e.g., C20-C26) often are activated and/or transported bythe thioesterification activity of a long-chain acyl-CoA synthetase(e.g., FAT1; EC 6.2.1.3; also referred to as long-chain fatty acid-CoAligase, acyl-CoA synthetase; fatty acid thiokinase (long chain);acyl-activating enzyme; palmitoyl-CoA synthase; lignoceroyl-CoAsynthase; arachidonyl-CoA synthetase; acyl coenzyme A synthetase;acyl-CoA ligase; palmitoyl coenzyme A synthetase; thiokinase;palmitoyl-CoA ligase; acyl-coenzyme A ligase; fatty acid CoA ligase;long-chain fatty acyl coenzyme A synthetase; oleoyl-CoA synthetase;stearoyl-CoA synthetase; long chain fatty acyl-CoA synthetase;long-chain acyl CoA synthetase; fatty acid elongase (ELO); LCFAsynthetase; pristanoyl-CoA synthetase; ACS3; long-chain acyl-CoAsynthetase I; long-chain acyl-CoA synthetase II; fatty acyl-coenzyme Asynthetase; long-chain acyl-coenzyme A synthetase; and acid:CoA ligase(AMP-forming)), in some organisms. Fatty acids also can be transportedinto the host organism from feedstocks by the activity of long chainacyl-CoA synthetase.

Long-chain acyl-CoA synthetase catalyzes the reaction,

ATP+a long-chain carboxylic acid+CoA=AMP+diphosphate+an acyl-CoA,

where “an acyl-CoA” refers to a fatty-acyl-CoA molecule. As notedherein, activation of fatty acids is often necessary for entry of fattyacids into various cellular processes (e.g., as an energy source, as acomponent for membrane formation and/or remodeling, as carbon storagemolecules). Deletion mutants of FAT1 have been shown to accumulate verylong chain fatty acids and exhibit decreased activation of these fattyacids. Without being limited by theory, it is believed that reduction inthe activity of long-chain acyl-CoA synthetase may reduce the amount oflong chain fatty acids converted into fatty-acyl-CoA intermediates,thereby increasing the amount of fatty acids available for conversioninto a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioicacid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) by other engineered pathwaysin the same host organism (e.g., omega oxidation pathway, beta oxidationpathway, omega oxidation pathway and beta oxidation pathway).Long-chain-acyl-CoA synthetase activity can be reduced or inactivated byany suitable means. Described herein are gene knockout methods suitablefor disrupting the nucleotide sequence that encodes the polypeptidehaving FAT1 activity. The nucleotide sequence of FAT1 is provided inExample 33, SEQ ID NO: 41. DNA vectors suitable for use in constructing“knockout” constructs are described herein.

The presence, absence or amount of long-chain-acyl-CoA synthetaseactivity can be detected by any suitable method known in the art.Non-limiting examples of suitable detection methods include enzymaticassays, binding assays (e.g., Erland et al, Analytical Biochemistry295(1):38-44 (2001)), PCR based assays (e.g., qPCR, RTPCR),immunological detection methods (e.g., antibodies specific forlong-chain-acyl-CoA synthetase), the like and combinations thereof.

Selective Modification of Fat1p to Retain Transport Activity

Transport of free fatty acids across a cellular membrane can occur bypassive diffusion or by protein-mediated active transport. The mechanismof passive diffusion can be manipulated (increased rate or decreasedrate) to some extent, by the choice of fatty acid feedstock or bychanging the extracellular environment. The rate of active transport offree fatty acids into the cell may be increased by amplifying transportproteins involved in fatty acid import. One such enzyme is Fat1p (e.g.,Fat1p of S. cerevisiae) which is a dual function protein with both fattyacid transport and acyl-CoA synthetase activities.

Discussed above were the benefits of knocking out enzymes with acyl-CoAsynthetase activity. In order to increase fatty acid transport into thecell without also increasing ACS activity, mutants of the Fat1p can beconstructed that are transport competent but ACS incompetent.

Fat1p can transport a free fatty acid across a cellular membrane and“activate” the fatty acid to an acyl-CoA thioester on the inner side ofthe cellular membrane. Once converted to an acyl-CoA thioester a fattyacid can enter the following biochemical pathways: 1) peroxisomalbeta-oxidation, 2) triacylglyceride synthesis, 3) cholesteryl estersynthesis or 4) phospholipid synthesis. All of these possible fates forfatty acyl-CoA can prevent the metabolism of an imported fatty acid intoa dicarboxylic acid and result in a low yield production of dicarboxylicacids from fatty acid feedstocks. Therefore, in some embodiments,strains are being developed with a mutant Fat1p enzyme (i.e.,Fat1p-mut), that retains fatty acid transport activity (e.g., theability to transport fatty acids across the cellular membrane) but lacksa thioesterase activity (e.g., the ability to activate a fatty acid toan acyl-CoA thioester). Fat1p mutants have been described in theliterature for S. cerevisiae. Some such mutants are known in theliterature and correspond to the mutants S244A or D495A in the Candidastrain ATCC20336 Fat1p enzyme.

Knocking Out Transport into the Peroxisome

The mechanism of transport of fatty acids into the peroxisome differsbased upon the chain length of the fatty acid. Long chain fatty acids,C16-C18, are not able to diffuse across the peroxisomal membrane in freeacid form but are instead transported across as fatty acyl-CoA esters inan ATP-dependent process catalyzed by the Pxa1p/Pxa2p heterodimer (e.g.EC 3.6.3.47). Short and medium chain fatty acids, C6-C14, are thought tobe able to diffuse across the peroxisomal membrane in the free acidform, however may also by aided in transport into the peroxisomal matrixby Pex11p or by other as yet unknown transporters. In some embodiments,knocking out the genes encoding these transport proteins would againimprove diacid yields by redirecting exogenously supplied fatty acidsfrom biochemical use in the peroxisome to the w-oxidation pathway.

Examples of the sequences of Pxa1p, Pxa2p and Pex11p from Candida strainATCC20336 are shown below.

-   Candida strain ATCC20336_PXA1—SEQ ID NO: 92-   Candida strain ATCC20336_PXA2—SEQ ID NO: 94-   Candida strain ATCC20336_PEX11—SEQ ID NO: 96

Acyl-CoA Sterol Acyltransferase

The term “a genetic modification that results in increased fatty acidsynthesis” as used herein also refers to a genetic alteration of a hostmicroorganism that reduces an endogenous activity that converts fattyacids into cholesterol esters. In some embodiments, an endogenousactivity that converts fatty acids into cholesterol esters is reduced.In certain embodiments, an acyl-CoA sterol acyltransferase activity isreduced. Such alterations can advantageously increase yields of endproducts, such as a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid).

Fatty acids can be converted into a cholesterol-ester by the activity ofacyl-CoA sterol acyltransferase (e.g., ARE1, ARE2, EC 2.3.1.26; alsoreferred to as sterol O-acyltransferase; cholesterol acyltransferase;sterol-ester synthase; sterol-ester synthetase; sterol-ester synthase;acyl coenzyme A-cholesterol-O-acyltransferase; acyl-CoA:cholesterolacyltransferase; ACAT; acylcoenzyme A:cholesterol O-acyltransferase;cholesterol ester synthase; cholesterol ester synthetase; andcholesteryl ester synthetase), in many organisms. Without being limitedby any theory, cholesterol esterification may be involved in directingfatty acids away from incorporation into cell membranes and towardsstorage forms of lipids. Acyl-CoA sterol acyltransferase catalyzes thereaction,

acyl-CoA+cholesterol=CoA+cholesterol ester.

The esterification of cholesterol is believed to limit its solubility incell membrane lipids and thus promotes accumulation of cholesterol esterin the fat droplets (e.g., a form of carbon storage molecule) withincytoplasm. Therefore, without being limited by any theory esterificationof cholesterol may cause the accumulation of lipid storage molecules,and disruption of the activity of acyl-CoA sterol acyltransferase maycause an increase in acyl-CoA levels that can be converted into a fattydicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) by other engineered pathwaysin the same host organism (e.g., omega oxidation pathway, beta oxidationpathway, omega oxidation pathway and beta oxidation pathway). Acyl-CoAsterol acyltransferase can be inactivated by any suitable means.Described herein are gene knockout methods suitable for disruptingnucleotide sequences that encode polypeptides having ARE1 activity, ARE2activity or ARE1 activity and ARE2 activity. The nucleotide sequences ofARE1 and ARE2 are provided in Example 33, SEQ ID NOS: 43 and 45. DNAvectors suitable for use in constructing “knockout” constructs aredescribed herein.

The presence, absence or amount of acyl-CoA sterol acyltransferaseactivity can be detected by any suitable method known in the art.Non-limiting examples of suitable detection methods include enzymaticassays (e.g., Chen et al, Plant Physiology 145:974-984 (2007)), bindingassays, PCR based assays (e.g., qPCR, RT-PCR), immunological detectionmethods (e.g., antibodies specific for long-chain-acyl-CoA synthetase),the like and combinations thereof.

Diacylglycerol Acyltransferase & Acyltransferases

The term “a genetic modification that results in increased fatty acidsynthesis” as used herein also refers to a genetic alteration of a hostmicroorganism that reduces an endogenous activity that catalyzesdiacylglycerol esterification (e.g., addition of acyl group to adiacylglycerol to form a triacylglycerol). In some embodiments, anendogenous activity that converts diacylglycerol into triacylglycerol isreduced. In certain embodiments, an acyltransferase activity is reduced.In some embodiments a diacylglycerol acyltransferase activity isreduced. In some embodiments a diacylglycerol acyltransferase (e.g.,DGA1, EC 2.3.1.20) activity and an acyltransferase (e.g., LRO1) activityare reduced. Such alterations can advantageously increase yields of endproducts, such as a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid).

Diacylglycerol can be converted into triacylglycerol by the activity ofdiacylglycerol acyltransferase (e.g., DGA1; EC 2.3.1.20; also referredto as diglyceride acyltransferase; 1,2-diacylglycerol acyltransferase;diacylglycerol acyltransferase; diglyceride O-acyltransferase;palmitoyl-CoA-sn-1,2-diacylglycerol acyltransferase;acyl-CoA:1,2-diacylglycerol O-acyltransferase andacyl-CoA:1,2-diacyl-sn-glycerol O-acyltransferase), in many organisms.Diacylglycerol acyltransferase catalyzes the reaction,

Acyl-CoA+1,2-diacyl-sn-glycerol=CoA+triacylglycerol,

and is generally considered the terminal and only committed step intriglyceride synthesis. The product of the DGA1 gene in yeast normallyis localized to lipid particles.

In addition to the diacylglycerol esterification activity described forDGA1, many organisms also can generate triglycerides by the activity ofother acyltransferase activities, non-limiting examples of which includelecithin-cholesterol acyltransferase activity (e.g., LRO1; EC 2.3.1.43;also referred to as phosphatidylcholine-sterol O-acyltransferaseactivity; lecithin-cholesterol acyltransferase activity;phospholipid-cholesterol acyltransferase activity; LCAT(lecithin-cholesterol acyltransferase) activity; lecithin:cholesterolacyltransferase activity; and lysolecithin acyltransferase activity) andphospholipid:diacylglycerol acyltransferase (e.g., EC 2.3.1.158; alsoreferred to as PDAT activity and phospholipid:1,2-diacyl-sn-glycerolO-acyltransferase activity). Acyltransferases of the families EC2.3.1.43 and EC 2.3.1.58 catalyze the general reaction,

phospholipid+1,2-diacylglycerol=lysophospholipid+triacylglycerol.

Triacylglycerides often are utilized as carbon (e.g., fatty acid orlipid) storage molecules. Without being limited by any theory, it isbelieve that reducing the activity of acyltransferase may reduce theconversion of diacylglycerol to triacylglycerol, which may causeincreased accumulation of fatty acid, in conjunction with additionalgenetic modifications (e.g., lipase to further remove fatty acids fromthe glycerol backbone) that can be converted into a fatty dicarboxylicacid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid) by other engineered pathways in the same hostorganism (e.g., omega oxidation pathway, beta oxidation pathway, omegaoxidation pathway and beta oxidation pathway). Acyltransferases can beinactivated by any suitable means. Described herein are gene knockoutmethods suitable for disrupting nucleotide sequences that encodepolypeptides having DGA1 activity, LRO1 activity or DGA1 activity andLRO1 activity. The nucleotide sequence of DGA1 is provided in Example33, SEQ ID NO: 47 The nucleotide sequence of LRO1 is provided in Example33, SEQ ID NO: 49. DNA vectors suitable for use in constructing“knockout” constructs are described herein.

The genes ARE1 and ARE2 in S. cerevisiae are also involved intriacylglyceride synthesis. Knocking out genes encoding these enzymescan redirect exogenously supplied fatty acids to ω-oxidation.

The presence, absence or amount of acyltransferase activity can bedetected by any suitable method known in the art. Non-limiting examplesof suitable detection methods include enzymatic assays (e.g., Geelen,Analytical Biochemistry 322(2):264-268 (2003), Dahlqvist et al, PNAS97(12):6487-6492 (2000)), binding assays, PCR based assays (e.g., qPCR,RTPCR), immunological detection methods (e.g., antibodies specific for aDGA1 or LRO1 acyltransferase), the like and combinations thereof.

Carnitine Acetyltransferase

Carnitine acetyltransferase (i.e., Cat2, Cat2p) is an enzyme targeted toboth the peroxisomal and mitochondrial compartments. It catalyzes thetransfer of an acetyl group from a CoA group to a carnitine (or viceversa depending on location) as shown below.

Acetyl-CoA+carnitine→acetylcarnitine+CoA

An acetyl-carnitine molecule is transported across the peroxisomal andmitochondrial membranes whereas an acetyl-CoA molecule is not. Thereforethe action of Cat2 (e.g., 2.3.1.7) provides one of three possible routesfor acetyl groups to leave the peroxisome. Acetyl-CoA produced bybeta-oxidation can be converted to acetyl-carnitine and transported tothe mitochondria for entry into the TCA cycle. In some embodiments, theactivity of Cat2 is decreased or eliminated in order to slow the exit ofacetyl-CoA from the peroxisome. In some embodiments, providing abottle-neck downstream of the adipic acid intermediate that is derivedfrom beta-oxidation is a strategy for improving the yield of adipicacid.

Carnitine O-acyltransferase

Carnitine O-acyltransferase (i.e., CROT, e.g., 2.3.1.137) is aperoxisomal enzyme that can transfer an acyl chain from a CoA group to acarnitine group as shown below.

Acyl-CoA+carnitine→acylcarnitine+CoA

The acyl-carnitine produced may then be transported out of theperoxisome for use elsewhere in the cell. The enzyme may act on acylchains of different chain lengths but is most active on short chains(C6-C8). Diacids that are prematurely pulled out of beta-oxidation andsent to other cellular compartments can represent a yield loss.

UDP-Glucosyltransferase

The UDP-glucosyltransferase enzyme (i.e., UGTA1, UgtA1p, e.g., 2.4.1.-)performs the first reaction in the synthesis of sophorolipids.Sophorolipids are a class of biosurfactant molecules produced by someyeast when exposed to hydrophobic environments. They are made up ofsophorose (2-O-β-D-glucopyranosyl-D-glucopyranose) attached through itsanomeric carbon to an ω- or (ω-1)-hydroxylated fatty acid of 16 or 18carbons. The most well-known yeast for producing sophorolipids isCandida bombicola. The pathway for sophorolipid production in this yeastproceeds via a step-wise transfer of two glucose molecules to ahydroxy-fatty acid. The first step is carried out by UgtA1p and thesecond step by UgtB1p (Saerens K M J, Roelants S L K W, VanBogaert I NA, Soetaert W (2011) FEMS Yeast Res 11: 123-132; Saerens K M J, Zhang J,Saey L, VanBogaert I N A, Soetaert W (2011) Yeast 28: 279-292). Thestepwise transfer of glucose from UDP-glucose to the w-end of thehydroxyl-fatty acid could represent a yield loss if w-hydroxy fattyacids produced in the first step of w-oxidation are pulled intosophorolipid production rather than diacid production.

Elongase(s)

“Elongase(s)” means those enzyme(s) in an organism that have ability to(i) extend the chain length of fatty acyl-CoA molecules, as for exampleconverting C-12 to C-16 fatty acyl-CoA molecules to C16-C18 fatty acids;(ii) elongate palmitoyl-CoA and stearoyl-CoA up to about 22 carbon fattyacids; or (iii) synthesize longer chain carbon fatty acids from shorterchain CoA primers such as C-18-CoA. In some embodiments, the expressionof an elongase is decreased or knocked out in a fatty dicarboxylic acidproducing yeast.

Polynucleotides and Polypeptides

A nucleic acid (e.g., also referred to herein as nucleic acid reagent,target nucleic acid, target nucleotide sequence, nucleic acid sequenceof interest or nucleic acid region of interest) can be from any sourceor composition, such as DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (shortinhibitory RNA), RNAi, tRNA or mRNA, for example, and can be in any form(e.g., linear, circular, supercoiled, single-stranded, double-stranded,and the like). A nucleic acid can also comprise DNA or RNA analogs(e.g., containing base analogs, sugar analogs and/or a non-nativebackbone and the like). It is understood that the term “nucleic acid”does not refer to or infer a specific length of the polynucleotidechain, thus polynucleotides and oligonucleotides are also included inthe definition. Deoxyribonucleotides include deoxyadenosine,deoxycytidine, deoxyguanosine and deoxythymidine. For RNA, the uracilbase is uridine.

A nucleic acid sometimes is a plasmid, phage, autonomously replicatingsequence (ARS), centromere, artificial chromosome, yeast artificialchromosome (e.g., YAC) or other nucleic acid able to replicate or bereplicated in a host cell. In certain embodiments a nucleic acid can befrom a library or can be obtained from enzymatically digested, shearedor sonicated genomic DNA (e.g., fragmented) from an organism ofinterest. In some embodiments, nucleic acid subjected to fragmentationor cleavage may have a nominal, average or mean length of about 5 toabout 10,000 base pairs, about 100 to about 1,000 base pairs, about 100to about 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000base pairs. Fragments can be generated by any suitable method in theart, and the average, mean or nominal length of nucleic acid fragmentscan be controlled by selecting an appropriate fragment-generatingprocedure by the person of ordinary skill. In some embodiments, thefragmented DNA can be size selected to obtain nucleic acid fragments ofa particular size range.

Nucleic acid can be fragmented by various methods known to the person ofordinary skill, which include without limitation, physical, chemical andenzymic processes. Examples of such processes are described in U.S.Patent Application Publication No. 20050112590 (published on May 26,2005, entitled “Fragmentation-based methods and systems for sequencevariation detection and discovery,” naming Van Den Boom et al.). Certainprocesses can be selected by the person of ordinary skill to generatenon-specifically cleaved fragments or specifically cleaved fragments.Examples of processes that can generate non-specifically cleavedfragment sample nucleic acid include, without limitation, contactingsample nucleic acid with apparatus that expose nucleic acid to shearingforce (e.g., passing nucleic acid through a syringe needle; use of aFrench press); exposing sample nucleic acid to irradiation (e.g., gamma,x-ray, UV irradiation; fragment sizes can be controlled by irradiationintensity); boiling nucleic acid in water (e.g., yields about 500 basepair fragments) and exposing nucleic acid to an acid and base hydrolysisprocess.

Nucleic acid may be specifically cleaved by contacting the nucleic acidwith one or more specific cleavage agents. The term “specific cleavageagent” as used herein refers to an agent, sometimes a chemical or anenzyme that can cleave a nucleic acid at one or more specific sites.Specific cleavage agents often will cleave specifically according to aparticular nucleotide sequence at a particular site. Examples of enzymicspecific cleavage agents include without limitation endonucleases (e.g.,DNase (e.g., DNase I, II); RNase (e.g., RNase E, F, H, P); Cleavase™enzyme; Taq DNA polymerase; E. coli DNA polymerase I and eukaryoticstructure-specific endonucleases; murine FEN-1 endonucleases; type I, IIor III restriction endonucleases such as Acc I, Afl III, Alu I, Alw44 I,Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl I. Bgl II, BlnI, Bsm I, BssH II, BstE II, Cfo I, Cla I, Dde I, Dpn I, Dra I, EcIX I,EcoR I, EcoR I, EcoR II, EcoR V, Hae II, Hae II, Hind II, Hind III, HpaI, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, Msp I, Nci I, Nco I, Nde I, NdeII, Nhe I, Not I, Nru I, Nsi I, Pst I, Pvu I, Pvu II, Rsa I, Sac I, SalI, Sau3A I, Sca I, ScrF I, Sfi I, Sma I, Spe I, Sph I, Ssp I, Stu I, StyI, Swa I, Taq I, Xba I, Xho I); glycosylases (e.g., uracil-DNAglycolsylase (UDG), 3-methyladenine DNA glycosylase, 3-methyladenine DNAglycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNAglycosylase, thymine mismatch-DNA glycosylase, hypoxanthine-DNAglycosylase, 5-Hydroxymethyluracil DNA glycosylase (HmUDG),5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNAglycosylase); exonucleases (e.g., exonuclease III); ribozymes, andDNAzymes. Sample nucleic acid may be treated with a chemical agent, orsynthesized using modified nucleotides, and the modified nucleic acidmay be cleaved. In non-limiting examples, sample nucleic acid may betreated with (i) alkylating agents such as methylnitrosourea thatgenerate several alkylated bases, including N3-methyladenine andN3-methylguanine, which are recognized and cleaved by alkyl purineDNA-glycosylase; (ii) sodium bisulfite, which causes deamination ofcytosine residues in DNA to form uracil residues that can be cleaved byuracil N-glycosylase; and (iii) a chemical agent that converts guanineto its oxidized form, 8-hydroxyguanine, which can be cleaved byformamidopyrimidine DNA N-glycosylase. Examples of chemical cleavageprocesses include without limitation alkylation, (e.g., alkylation ofphosphorothioate-modified nucleic acid); cleavage of acid lability ofP3′-N5′-phosphoroamidate-containing nucleic acid; and osmium tetroxideand piperidine treatment of nucleic acid.

As used herein, the term “complementary cleavage reactions” refers tocleavage reactions that are carried out on the same nucleic acid usingdifferent cleavage reagents or by altering the cleavage specificity ofthe same cleavage reagent such that alternate cleavage patterns of thesame target or reference nucleic acid or protein are generated. Incertain embodiments, nucleic acids of interest may be treated with oneor more specific cleavage agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore specific cleavage agents) in one or more reaction vessels (e.g.,nucleic acid of interest is treated with each specific cleavage agent ina separate vessel).

A nucleic acid suitable for use in the embodiments described hereinsometimes is amplified by any amplification process known in the art(e.g., PCR, RT-PCR and the like). Nucleic acid amplification may beparticularly beneficial when using organisms that are typicallydifficult to culture (e.g., slow growing, require specialize cultureconditions and the like). The terms “amplify”, “amplification”,“amplification reaction”, or “amplifying” as used herein refer to any invitro processes for multiplying the copies of a target sequence ofnucleic acid. Amplification sometimes refers to an “exponential”increase in target nucleic acid. However, “amplifying” as used hereincan also refer to linear increases in the numbers of a select targetsequence of nucleic acid, but is different than a one-time, singleprimer extension step. In some embodiments, a limited amplificationreaction, also known as pre-amplification, can be performed.Pre-amplification is a method in which a limited amount of amplificationoccurs due to a small number of cycles, for example 10 cycles, beingperformed. Pre-amplification can allow some amplification, but stopsamplification prior to the exponential phase, and typically producesabout 500 copies of the desired nucleotide sequence(s). Use ofpre-amplification may also limit inaccuracies associated with depletedreactants in standard PCR reactions.

In some embodiments, a nucleic acid reagent sometimes is stablyintegrated into the chromosome of the host organism, or a nucleic acidreagent can be a deletion of a portion of the host chromosome, incertain embodiments (e.g., genetically modified organisms, wherealteration of the host genome confers the ability to selectively orpreferentially maintain the desired organism carrying the geneticmodification). Such nucleic acid reagents (e.g., nucleic acids orgenetically modified organisms whose altered genome confers a selectabletrait to the organism) can be selected for their ability to guideproduction of a desired protein or nucleic acid molecule. When desired,the nucleic acid reagent can be altered such that codons encode for (i)the same amino acid, using a different tRNA than that specified in thenative sequence, or (ii) a different amino acid than is normal,including unconventional or unnatural amino acids (including detectablylabeled amino acids). As described herein, the term “native sequence”refers to an unmodified nucleotide sequence as found in its naturalsetting (e.g., a nucleotide sequence as found in an organism).

A nucleic acid or nucleic acid reagent can comprise certain elementsoften selected according to the intended use of the nucleic acid. Any ofthe following elements can be included in or excluded from a nucleicacid reagent. A nucleic acid reagent, for example, may include one ormore or all of the following nucleotide elements: one or more promoterelements, one or more 5′ untranslated regions (5′UTRs), one or moreregions into which a target nucleotide sequence may be inserted (an“insertion element”), one or more target nucleotide sequences, one ormore 3′ untranslated regions (3′UTRs), and one or more selectionelements. A nucleic acid reagent can be provided with one or more ofsuch elements and other elements may be inserted into the nucleic acidbefore the nucleic acid is introduced into the desired organism. In someembodiments, a provided nucleic acid reagent comprises a promoter,5′UTR, optional 3′UTR and insertion element(s) by which a targetnucleotide sequence is inserted (i.e., cloned) into the nucleotide acidreagent. In certain embodiments, a provided nucleic acid reagentcomprises a promoter, insertion element(s) and optional 3′UTR, and a 5′UTR/target nucleotide sequence is inserted with an optional 3′UTR. Theelements can be arranged in any order suitable for expression in thechosen expression system (e.g., expression in a chosen organism, orexpression in a cell free system, for example), and in some embodimentsa nucleic acid reagent comprises the following elements in the 5′ to 3′direction: (1) promoter element, 5′UTR, and insertion element(s); (2)promoter element, 5′UTR, and target nucleotide sequence; (3) promoterelement, 5′UTR, insertion element(s) and 3′UTR; and (4) promoterelement, 5′UTR, target nucleotide sequence and 3′UTR.

Promoters

A promoter element typically is required for DNA synthesis and/or RNAsynthesis. A promoter element often comprises a region of DNA that canfacilitate the transcription of a particular gene, by providing a startsite for the synthesis of RNA corresponding to a gene. Promotersgenerally are located near the genes they regulate, are located upstreamof the gene (e.g., 5′ of the gene), and are on the same strand of DNA asthe sense strand of the gene, in some embodiments. In some embodiments,a promoter element can be isolated from a gene or organism and insertedin functional connection with a polynucleotide sequence to allow alteredand/or regulated expression. A non-native promoter (e.g., promoter notnormally associated with a given nucleic acid sequence) used forexpression of a nucleic acid often is referred to as a heterologouspromoter. In certain embodiments, a heterologous promoter and/or a 5′UTRcan be inserted in functional connection with a polynucleotide thatencodes a polypeptide having a desired activity as described herein. Theterms “operably linked” and “in functional connection with” as usedherein with respect to promoters, refer to a relationship between acoding sequence and a promoter element. The promoter is operably linkedor in functional connection with the coding sequence when expressionfrom the coding sequence via transcription is regulated, or controlledby, the promoter element. The terms “operably linked” and “in functionalconnection with” are utilized interchangeably herein with respect topromoter elements.

A promoter often interacts with a RNA polymerase. A polymerase is anenzyme that catalyses synthesis of nucleic acids using a preexistingnucleic acid reagent. When the template is a DNA template, an RNAmolecule is transcribed before protein is synthesized. Enzymes havingpolymerase activity suitable for use in the present methods include anypolymerase that is active in the chosen system with the chosen templateto synthesize protein. In some embodiments, a promoter (e.g., aheterologous promoter) also referred to herein as a promoter element,can be operably linked to a nucleotide sequence or an open reading frame(ORF).

Transcription from the promoter element can catalyze the synthesis of anRNA corresponding to the nucleotide sequence or ORF sequence operablylinked to the promoter, which in turn leads to synthesis of a desiredpeptide, polypeptide or protein.

Promoter elements sometimes exhibit responsiveness to regulatorycontrol. Promoter elements also sometimes can be regulated by aselective agent. That is, transcription from promoter elements sometimescan be turned on, turned off, up-regulated or down-regulated, inresponse to a change in environmental, nutritional or internalconditions or signals (e.g., heat inducible promoters, light regulatedpromoters, feedback regulated promoters, hormone influenced promoters,tissue specific promoters, oxygen and pH influenced promoters, promotersthat are responsive to selective agents (e.g., kanamycin) and the like,for example). Promoters influenced by environmental, nutritional orinternal signals frequently are influenced by a signal (direct orindirect) that binds at or near the promoter and increases or decreasesexpression of the target sequence under certain conditions.

Non-limiting examples of selective or regulatory agents that caninfluence transcription from a promoter element used in embodimentsdescribed herein include, without limitation, (1) nucleic acid segmentsthat encode products that provide resistance against otherwise toxiccompounds (e.g., antibiotics); (2) nucleic acid segments that encodeproducts that are otherwise lacking in the recipient cell (e.g.,essential products, tRNA genes, auxotrophic markers); (3) nucleic acidsegments that encode products that suppress the activity of a geneproduct; (4) nucleic acid segments that encode products that can bereadily identified (e.g., phenotypic markers such as antibiotics (e.g.,β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), and cell surface proteins); (5) nucleic acidsegments that bind products that are otherwise detrimental to cellsurvival and/or function; (6) nucleic acid segments that otherwiseinhibit the activity of any of the nucleic acid segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acidsegments that bind products that modify a substrate (e.g., restrictionendonucleases); (8) nucleic acid segments that can be used to isolate oridentify a desired molecule (e.g., specific protein binding sites); (9)nucleic acid segments that encode a specific nucleotide sequence thatcan be otherwise non-functional (e.g., for PCR amplification ofsubpopulations of molecules); (10) nucleic acid segments that, whenabsent, directly or indirectly confer resistance or sensitivity toparticular compounds; (11) nucleic acid segments that encode productsthat either are toxic or convert a relatively non-toxic compound to atoxic compound (e.g., Herpes simplex thymidine kinase, cytosinedeaminase) in recipient cells; (12) nucleic acid segments that inhibitreplication, partition or heritability of nucleic acid molecules thatcontain them; and/or (13) nucleic acid segments that encode conditionalreplication functions, e.g., replication in certain hosts or host cellstrains or under certain environmental conditions (e.g., temperature,nutritional conditions, and the like). In some embodiments, theregulatory or selective agent can be added to change the existing growthconditions to which the organism is subjected (e.g., growth in liquidculture, growth in a fermentor, growth on solid nutrient plates and thelike for example).

In some embodiments, regulation of a promoter element can be used toalter (e.g., increase, add, decrease or substantially eliminate) theactivity of a peptide, polypeptide or protein (e.g., enzyme activity forexample). For example, a microorganism can be engineered by geneticmodification to express a nucleic acid reagent that can add a novelactivity (e.g., an activity not normally found in the host organism) orincrease the expression of an existing activity by increasingtranscription from a homologous or heterologous promoter operably linkedto a nucleotide sequence of interest (e.g., homologous or heterologousnucleotide sequence of interest), in certain embodiments. In someembodiments, a microorganism can be engineered by genetic modificationto express a nucleic acid reagent that can decrease expression of anactivity by decreasing or substantially eliminating transcription from ahomologous or heterologous promoter operably linked to a nucleotidesequence of interest, in certain embodiments.

In some embodiments the activity can be altered using recombinant DNAand genetic techniques known to the artisan. Methods for engineeringmicroorganisms are further described herein. Tables herein providenon-limiting lists of yeast promoters that are up-regulated by oxygen,yeast promoters that are down-regulated by oxygen, yeast transcriptionalrepressors and their associated genes, DNA binding motifs as determinedusing the MEME sequence analysis software. Potential regulator bindingmotifs can be identified using the program MEME to search intergenicregions bound by regulators for overrepresented sequences. For eachregulator, the sequences of intergenic regions bound with p-values lessthan 0.001 were extracted to use as input for motif discovery. The MEMEsoftware was run using the following settings: a motif width rangingfrom 6 to 18 bases, the “zoops” distribution model, a 6^(th) orderMarkov background model and a discovery limit of 20 motifs. Thediscovered sequence motifs were scored for significance by two criteria:an E-value calculated by MEME and a specificity score. The motif withthe best score using each metric is shown for each regulator. All motifspresented are derived from datasets generated in rich growth conditionswith the exception of a previously published dataset for epitope-taggedGal4 grown in galactose.

In some embodiments, the altered activity can be found by screening theorganism under conditions that select for the desired change inactivity. For example, certain microorganisms can be adapted to increaseor decrease an activity by selecting or screening the organism inquestion on a media containing substances that are poorly metabolized oreven toxic. An increase in the ability of an organism to grow asubstance that is normally poorly metabolized may result in an increasein the growth rate on that substance, for example. A decrease in thesensitivity to a toxic substance might be manifested by growth on higherconcentrations of the toxic substance, for example. Geneticmodifications that are identified in this manner sometimes are referredto as naturally occurring mutations or the organisms that carry them cansometimes be referred to as naturally occurring mutants. Modificationsobtained in this manner are not limited to alterations in promotersequences. That is, screening microorganisms by selective pressure, asdescribed above, can yield genetic alterations that can occur innon-promoter sequences, and sometimes also can occur in sequences thatare not in the nucleotide sequence of interest, but in a relatednucleotide sequences (e.g., a gene involved in a different step of thesame pathway, a transport gene, and the like). Naturally occurringmutants sometimes can be found by isolating naturally occurring variantsfrom unique environments, in some embodiments.

Homology and Identity

In addition to the regulated promoter sequences, regulatory sequences,and coding polynucleotides provided herein, a nucleic acid reagent mayinclude a polynucleotide sequence 80% or more identical to the foregoing(or to the complementary sequences). That is, a nucleotide sequence thatis at least 80% or more, 81% or more, 82% or more, 83% or more, 84% ormore, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more,90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% ormore, 96% or more, 97% or more, 98% or more, or 99% or more identical toa nucleotide sequence described herein can be utilized. The term“identical” as used herein refers to two or more nucleotide sequenceshaving substantially the same nucleotide sequence when compared to eachother. One test for determining whether two nucleotide sequences oramino acids sequences are substantially identical is to determine thepercent of identical nucleotide sequences or amino acid sequencesshared.

Calculations of sequence identity can be performed as follows. Sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The length of a reference sequencealigned for comparison purposes is sometimes 30% or more, 40% or more,50% or more, often 60% or more, and more often 70% or more, 80% or more,90% or more, or 100% of the length of the reference sequence. Thenucleotides or amino acids at corresponding nucleotide or polypeptidepositions, respectively, are then compared among the two sequences. Whena position in the first sequence is occupied by the same nucleotide oramino acid as the corresponding position in the second sequence, thenucleotides or amino acids are deemed to be identical at that position.The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, introduced foroptimal alignment of the two sequences.

Comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm.Percent identity between two amino acid or nucleotide sequences can bedetermined using the algorithm of Meyers & Miller, CABIOS 4: 11-17(1989), which has been incorporated into the ALIGN program (version2.0), using a PAM120 weight residue table, a gap length penalty of 12and a gap penalty of 4. Also, percent identity between two amino acidsequences can be determined using the Needleman & Wunsch, J. Mol. Biol.48: 444-453 (1970) algorithm which has been incorporated into the GAPprogram in the GCG software package (available at the http addresswww.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and agap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3,4, 5, or 6. Percent identity between two nucleotide sequences can bedetermined using the GAP program in the GCG software package (availableat http address www.gcg.com), using a NWSgapdna.CMP matrix and a gapweight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or6. A set of parameters often used is a Blossum 62 scoring matrix with agap open penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

Sequence identity can also be determined by hybridization assaysconducted under stringent conditions. As use herein, the term “stringentconditions” refers to conditions for hybridization and washing.Stringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons,N.Y., 6.3.1-6.3.6 (1989). Aqueous and non-aqueous methods are describedin that reference and either can be used. An example of stringenthybridization conditions is hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions ishybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C. Often,stringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 65° C. More often, stringency conditionsare 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or morewashes at 0.2×SSC, 1% SDS at 65° C.

UTRs

As noted above, nucleic acid reagents may also comprise one or more 5′UTR's, and one or more 3′UTR's. A 5′ UTR may comprise one or moreelements endogenous to the nucleotide sequence from which it originates,and sometimes includes one or more exogenous elements. A 5′ UTR canoriginate from any suitable nucleic acid, such as genomic DNA, plasmidDNA, RNA or mRNA, for example, from any suitable organism (e.g., virus,bacterium, yeast, fungi, plant, insect or mammal). The artisan mayselect appropriate elements for the 5′ UTR based upon the chosenexpression system (e.g., expression in a chosen organism, or expressionin a cell free system, for example). A 5′ UTR sometimes comprises one ormore of the following elements known to the artisan: enhancer sequences(e.g., transcriptional or translational), transcription initiation site,transcription factor binding site, translation regulation site,translation initiation site, translation factor binding site, accessoryprotein binding site, feedback regulation agent binding sites, Pribnowbox, TATA box, −35 element, E-box (helix-loop-helix binding element),ribosome binding site, replicon, internal ribosome entry site (IRES),silencer element and the like. In some embodiments, a promoter elementmay be isolated such that all 5′ UTR elements necessary for properconditional regulation are contained in the promoter element fragment,or within a functional subsequence of a promoter element fragment.

A 5′UTR in the nucleic acid reagent can comprise a translationalenhancer nucleotide sequence. A translational enhancer nucleotidesequence often is located between the promoter and the target nucleotidesequence in a nucleic acid reagent. A translational enhancer sequenceoften binds to a ribosome, sometimes is an 18S rRNA-bindingribonucleotide sequence (i.e., a 40S ribosome binding sequence) andsometimes is an internal ribosome entry sequence (IRES). An IRESgenerally forms an RNA scaffold with precisely placed RNA tertiarystructures that contact a 40S ribosomal subunit via a number of specificintermolecular interactions. Examples of ribosomal enhancer sequencesare known and can be identified by the artisan (e.g., Mignone et al.,Nucleic Acids Research 33: 0141-D146 (2005); Paulous et al., NucleicAcids Research 31: 722-733 (2003); Akbergenov et al., Nucleic AcidsResearch 32: 239-247 (2004); Mignone et al., Genome Biology 3(3):reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:3401-3411 (2002); Shaloiko et al., http addresswww.interscience.wiley.com, DOI: 10.1002/bit.20267; and Gallie et al.,Nucleic Acids Research 15: 3257-3273 (1987)).

A translational enhancer sequence sometimes is a eukaryotic sequence,such as a Kozak consensus sequence or other sequence (e.g., hydroidpolyp sequence, GenBank accession no. U07128). A translational enhancersequence sometimes is a prokaryotic sequence, such as a Shine-Dalgarnoconsensus sequence. In certain embodiments, the translational enhancersequence is a viral nucleotide sequence. A translational enhancersequence sometimes is from a 5′ UTR of a plant virus, such as TobaccoMosaic Virus (TMV), Alfalfa Mosaic Virus (AMV); Tobacco Etch Virus(ETV); Potato Virus Y (PVY); Turnip Mosaic (poty) Virus and Pea SeedBorne Mosaic Virus, for example. In certain embodiments, an omegasequence about 67 bases in length from TMV is included in the nucleicacid reagent as a translational enhancer sequence (e.g., devoid ofguanosine nucleotides and includes a 25 nucleotide long poly (CAA)central region).

A 3′ UTR may comprise one or more elements endogenous to the nucleotidesequence from which it originates and sometimes includes one or moreexogenous elements. A 3′ UTR may originate from any suitable nucleicacid, such as genomic DNA, plasmid DNA, RNA or mRNA, for example, fromany suitable organism (e.g., a virus, bacterium, yeast, fungi, plant,insect or mammal). The artisan can select appropriate elements for the3′ UTR based upon the chosen expression system (e.g., expression in achosen organism, for example). A 3′ UTR sometimes comprises one or moreof the following elements known to the artisan: transcription regulationsite, transcription initiation site, transcription termination site,transcription factor binding site, translation regulation site,translation termination site, translation initiation site, translationfactor binding site, ribosome binding site, replicon, enhancer element,silencer element and polyadenosine tail. A 3′ UTR often includes apolyadenosine tail and sometimes does not, and if a polyadenosine tailis present, one or more adenosine moieties may be added or deleted fromit (e.g., about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45 or about 50 adenosine moieties may be addedor subtracted).

In some embodiments, modification of a 5′ UTR and/or a 3′ UTR can beused to alter (e.g., increase, add, decrease or substantially eliminate)the activity of a promoter. Alteration of the promoter activity can inturn alter the activity of a peptide, polypeptide or protein (e.g.,enzyme activity for example), by a change in transcription of thenucleotide sequence(s) of interest from an operably linked promoterelement comprising the modified 5′ or 3′ UTR. For example, amicroorganism can be engineered by genetic modification to express anucleic acid reagent comprising a modified 5′ or 3′ UTR that can add anovel activity (e.g., an activity not normally found in the hostorganism) or increase the expression of an existing activity byincreasing transcription from a homologous or heterologous promoteroperably linked to a nucleotide sequence of interest (e.g., homologousor heterologous nucleotide sequence of interest), in certainembodiments. In some embodiments, a microorganism can be engineered bygenetic modification to express a nucleic acid reagent comprising amodified 5′ or 3′ UTR that can decrease the expression of an activity bydecreasing or substantially eliminating transcription from a homologousor heterologous promoter operably linked to a nucleotide sequence ofinterest, in certain embodiments.

Target Nucleotide Sequence

A nucleotide reagent sometimes can comprise a target nucleotidesequence. A “target nucleotide sequence” as used herein encodes anucleic acid, peptide, polypeptide or protein of interest, and may be aribonucleotide sequence or a deoxyribonucleotide sequence. A targetnucleic acid sometimes is an untranslated ribonucleic acid and sometimesis a translated ribonucleic acid. An untranslated ribonucleic acid mayinclude, but is not limited to, a small interfering ribonucleic acid(siRNA), a short hairpin ribonucleic acid (shRNA), other ribonucleicacid capable of RNA interference (RNAi), an antisense ribonucleic acid,or a ribozyme. A translatable target nucleotide sequence (e.g., a targetribonucleotide sequence) sometimes encodes a peptide, polypeptide orprotein, which are sometimes referred to herein as “target peptides,”“target polypeptides” or “target proteins”.

Any peptides, polypeptides or proteins, or an activity catalyzed by oneor more peptides, polypeptides or proteins may be encoded by a targetnucleotide sequence and may be selected by a user. Representativeproteins include enzymes (e.g., acetyl-CoA carboxylase, acyl-CoAoxidase, thioesterase, monooxygenase, monooxygenase reductase, fattyalcohol oxidase, acyltransferase and the like, for example), antibodies,serum proteins (e.g., albumin), membrane bound proteins, hormones (e.g.,growth hormone, erythropoietin, insulin, etc.), cytokines, etc., andinclude both naturally occurring and exogenously expressed polypeptides.Representative activities (e.g., enzymes or combinations of enzymeswhich are functionally associated to provide an activity) includethioesterase activity, monooxygenase activity, monooxygenase reductaseactivity, acyltransferase activity, omega hydroxyl fatty aciddehydrogenase activity, beta-oxidation activity, omega-oxidationactivity and the like, for example. The term “enzyme” as used hereinrefers to a protein which can act as a catalyst to induce a chemicalchange in other compounds, thereby producing one or more products fromone or more substrates.

Specific polypeptides (e.g., enzymes) useful for embodiments describedherein are listed herein. The term “protein” as used herein refers to amolecule having a sequence of amino acids linked by peptide bonds. Thisterm includes fusion proteins, oligopeptides, peptides, cyclic peptides,polypeptides and polypeptide derivatives, whether native or recombinant,and also includes fragments, derivatives, homologs, and variantsthereof. A protein or polypeptide sometimes is of intracellular origin(e.g., located in the nucleus, cytosol, or interstitial space of hostcells in vivo) and sometimes is a cell membrane protein in vivo. In someembodiments (described above, and in further detail hereafter inEngineering and Alteration Methods), a genetic modification can resultin a modification (e.g., increase, substantially increase, decrease orsubstantially decrease) of a target activity.

A translatable nucleotide sequence generally is located between a startcodon (AUG in ribonucleic acids and ATG in deoxyribonucleic acids) and astop codon (e.g., UAA (ochre), UAG (amber) or UGA (opal) in ribonucleicacids and TAA, TAG or TGA in deoxyribonucleic acids), and sometimes isreferred to herein as an “open reading frame” (ORF). A translatablenucleotide sequence (e.g., ORF) sometimes is encoded differently in oneorganism (e.g., most organisms encode CTG as leucine) than in anotherorganism (e.g., C. tropicalis encodes CTG as serine). In someembodiments, a translatable nucleotide sequence is altered to correctalternate genetic code (e.g., codon usage) differences between anucleotide donor organism and an nucleotide recipient organism (e.g.,engineered organism). In certain embodiments, a translatable nucleotidesequence is altered to improve; (i) codon usage, (ii) transcriptionalefficiency, (iii) translational efficiency, (iv) the like, andcombinations thereof.

Nucleic Acid Reagents & Tools

A nucleic acid reagent sometimes comprises one or more ORFs. An ORF maybe from any suitable source, sometimes from genomic DNA, mRNA, reversetranscribed RNA or complementary DNA (cDNA) or a nucleic acid librarycomprising one or more of the foregoing, and is from any organismspecies that contains a nucleic acid sequence of interest, protein ofinterest, or activity of interest. Non-limiting examples of organismsfrom which an ORF can be obtained include bacteria, yeast, fungi, human,insect, nematode, bovine, equine, canine, feline, rat or mouse, forexample.

A nucleic acid reagent sometimes comprises a nucleotide sequenceadjacent to an ORF that is translated in conjunction with the ORF andencodes an amino acid tag. The tag-encoding nucleotide sequence islocated 3′ and/or 5′ of an ORF in the nucleic acid reagent, therebyencoding a tag at the C-terminus or N-terminus of the protein or peptideencoded by the ORF. Any tag that does not abrogate in vitrotranscription and/or translation may be utilized and may beappropriately selected by the artisan. Tags may facilitate isolationand/or purification of the desired ORF product from culture orfermentation media.

A tag sometimes specifically binds a molecule or moiety of a solid phaseor a detectable label, for example, thereby having utility forisolating, purifying and/or detecting a protein or peptide encoded bythe ORF. In some embodiments, a tag comprises one or more of thefollowing elements: FLAG (e.g., DYKDDDDKG), V5 (e.g., GKPIPNPLLGLDST),c-MYC (e.g., EQKLISEEDL), HSV (e.g., QPELAPEDPED), influenzahemaglutinin, HA (e.g., YPYDVPDYA), VSV-G (e.g., YTDIEMNRLGK), bacterialglutathione-S-transferase, maltose binding protein, a streptavidin- oravidin-binding tag (e.g., pcDNA™6 BioEase™ Gateway® Biotinylation System(Invitrogen)), thioredoxin, β-galactosidase, VSV-glycoprotein, afluorescent protein (e.g., green fluorescent protein or one of its manycolor variants (e.g., yellow, red, blue)), a polylysine or polyargininesequence, a polyhistidine sequence (e.g., His6) or other sequence thatchelates a metal (e.g., cobalt, zinc, copper), and/or a cysteine-richsequence that binds to an arsenic-containing molecule. In certainembodiments, a cysteine-rich tag comprises the amino acid sequenceCC-Xn-CC, wherein X is any amino acid and n is 1 to 3, and thecysteine-rich sequence sometimes is CCPGCC. In certain embodiments, thetag comprises a cysteine-rich element and a polyhistidine element (e.g.,CCPGCC and His6).

A tag often conveniently binds to a binding partner. For example, sometags bind to an antibody (e.g., FLAG) and sometimes specifically bind toa small molecule. For example, a polyhistidine tag specifically chelatesa bivalent metal, such as copper, zinc and cobalt; a polylysine orpolyarginine tag specifically binds to a zinc finger; a glutathioneS-transferase tag binds to glutathione; and a cysteine-rich tagspecifically binds to an arsenic-containing molecule. Arsenic-containingmolecules include LUMIO™ agents (Invitrogen, California), such as FlAsH™(EDT2[4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2])and ReAsH reagents (e.g., U.S. Pat. No. 5,932,474 to Tsien et al.,entitled “Target Sequences for Synthetic Molecules;” U.S. Pat. No.6,054,271 to Tsien et al., entitled “Methods of Using SyntheticMolecules and Target Sequences;” U.S. Pat. Nos. 6,451,569 and 6,008,378;published U.S. Patent Application 2003/0083373, and published PCT PatentApplication WO 99/21013, all to Tsien et al. and all entitled “SyntheticMolecules that Specifically React with Target Sequences”). Suchantibodies and small molecules sometimes are linked to a solid phase forconvenient isolation of the target protein or target peptide.

A tag sometimes comprises a sequence that localizes a translated proteinor peptide to a component in a system, which is referred to as a “signalsequence” or “localization signal sequence” herein. A signal sequenceoften is incorporated at the N-terminus of a target protein or targetpeptide, and sometimes is incorporated at the C-terminus. Examples ofsignal sequences are known to the artisan, are readily incorporated intoa nucleic acid reagent, and often are selected according to the organismin which expression of the nucleic acid reagent is performed. A signalsequence in some embodiments localizes a translated protein or peptideto a cell membrane. Examples of signal sequences include, but are notlimited to, a nucleus targeting signal (e.g., steroid receptor sequenceand N-terminal sequence of SV40 virus large T antigen); mitochondrialtargeting signal (e.g., amino acid sequence that forms an amphipathichelix); peroxisome targeting signal (e.g., C-terminal sequence in YFGfrom S. cerevisiae); and a secretion signal (e.g., N-terminal sequencesfrom invertase, mating factor alpha, PHO5 and SUC2 in S. cerevisiae;multiple N-terminal sequences of B. subtilis proteins (e.g., Tjalsma etal., Microbiol. Molec. Biol. Rev. 64: 515-547 (2000)); alpha amylasesignal sequence (e.g., U.S. Pat. No. 6,288,302); pectate lyase signalsequence (e.g., U.S. Pat. No. 5,846,818); precollagen signal sequence(e.g., U.S. Pat. No. 5,712,114); OmpA signal sequence (e.g., U.S. Pat.No. 5,470,719); lam beta signal sequence (e.g., U.S. Pat. No.5,389,529); B. brevis signal sequence (e.g., U.S. Pat. No. 5,232,841);and P. pastoris signal sequence (e.g., U.S. Pat. No. 5,268,273)).

A tag sometimes is directly adjacent to the amino acid sequence encodedby an ORF (i.e., there is no intervening sequence) and sometimes a tagis substantially adjacent to an ORF encoded amino acid sequence (e.g.,an intervening sequence is present). An intervening sequence sometimesincludes a recognition site for a protease, which is useful for cleavinga tag from a target protein or peptide. In some embodiments, theintervening sequence is cleaved by Factor Xa (e.g., recognition site I(E/D)GR), thrombin (e.g., recognition site LVPRGS), enterokinase (e.g.,recognition site DDDDK), TEV protease (e.g., recognition site ENLYFQG)or PreScission™ protease (e.g., recognition site LEVLFQGP), for example.

An intervening sequence sometimes is referred to herein as a “linkersequence,” and may be of any suitable length selected by the artisan. Alinker sequence sometimes is about 1 to about 20 amino acids in length,and sometimes about 5 to about 10 amino acids in length. The artisan mayselect the linker length to substantially preserve target protein orpeptide function (e.g., a tag may reduce target protein or peptidefunction unless separated by a linker), to enhance disassociation of atag from a target protein or peptide when a protease cleavage site ispresent (e.g., cleavage may be enhanced when a linker is present), andto enhance interaction of a tag/target protein product with a solidphase. A linker can be of any suitable amino acid content, and oftencomprises a higher proportion of amino acids having relatively shortside chains (e.g., glycine, alanine, serine and threonine).

A nucleic acid reagent sometimes includes a stop codon between a tagelement and an insertion element or ORF, which can be useful fortranslating an ORF with or without the tag. Mutant tRNA molecules thatrecognize stop codons (described above) suppress translation terminationand thereby are designated “suppressor tRNAs.” Suppressor tRNAs canresult in the insertion of amino acids and continuation of translationpast stop codons (e.g., U.S. Patent Application No. 60/587,583, filedJul. 14, 2004, entitled “Production of Fusion Proteins by Cell-FreeProtein Synthesis,”; Eggertsson, et al., (1988) Microbiological Review52(3):354-374, and Engleerg-Kukla, et al. (1996) in Escherichia coli andSalmonella Cellular and Molecular Biology, Chapter 60, pps 909-921,Neidhardt, et al. eds., ASM Press, Washington, D.C.). A number ofsuppressor tRNAs are known, including but not limited to, supE, supP,supD, supF and supZ suppressors, which suppress the termination oftranslation of the amber stop codon; supB, gIT, supL, supN, supC andsupM suppressors, which suppress the function of the ochre stop codonand glyT, trpT and Su-9 suppressors, which suppress the function of theopal stop codon. In general, suppressor tRNAs contain one or moremutations in the anti-codon loop of the tRNA that allows the tRNA tobase pair with a codon that ordinarily functions as a stop codon. Themutant tRNA is charged with its cognate amino acid residue and thecognate amino acid residue is inserted into the translating polypeptidewhen the stop codon is encountered. Mutations that enhance theefficiency of termination suppressors (i.e., increase stop codonread-through) have been identified. These include, but are not limitedto, mutations in the uar gene (also known as the prfA gene), mutationsin the ups gene, mutations in the sueA, sueB and sueC genes, mutationsin the rpsD (ramA) and rpsE (spcA) genes and mutations in the rplL gene.

Thus, a nucleic acid reagent comprising a stop codon located between anORF and a tag can yield a translated ORF alone when no suppressor tRNAis present in the translation system, and can yield a translated ORF-tagfusion when a suppressor tRNA is present in the system. Suppressor tRNAcan be generated in cells transfected with a nucleic acid encoding thetRNA (e.g., a replication incompetent adenovirus containing the humantRNA-Ser suppressor gene can be transfected into cells, or a YACcontaining a yeast or bacterial tRNA suppressor gene can be transfectedinto yeast cells, for example). Vectors for synthesizing suppressor tRNAand for translating ORFs with or without a tag are available to theartisan (e.g., Tag-On-Demand™kit (Invitrogen Corporation, California);Tag-On-Demand™ Suppressor Supernatant Instruction Manual, Version B, 6Jun. 2003, at http addresswww.invitrogen.com/content/sfs/manuals/tagondemand_supernatant_man.pdf;Tag-On-Demand™ Gateway® Vector Instruction Manual, Version B, 20 June,2003 at http addresswww.invitrogen.com/content/sfs/manuals/tagondemand_vectors_man.pdf; andCapone et al., Amber, ochre and opal suppressor tRNA genes derived froma human serine tRNA gene. EMBO J. 4:213, 1985).

Any convenient cloning strategy known in the art may be utilized toincorporate an element, such as an ORF, into a nucleic acid reagent.Known methods can be utilized to insert an element into the templateindependent of an insertion element, such as (1) cleaving the templateat one or more existing restriction enzyme sites and ligating an elementof interest and (2) adding restriction enzyme sites to the template byhybridizing oligonucleotide primers that include one or more suitablerestriction enzyme sites and amplifying by polymerase chain reaction(described in greater detail herein). Other cloning strategies takeadvantage of one or more insertion sites present or inserted into thenucleic acid reagent, such as an oligonucleotide primer hybridizationsite for PCR, for example, and others described herein. In someembodiments, a cloning strategy can be combined with geneticmanipulation such as recombination (e.g., recombination of a nucleicacid reagent with a nucleic acid sequence of interest into the genome ofthe organism to be modified, as described further herein). In someembodiments, the cloned ORF(s) can produce (directly or indirectly) afatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid), by engineering amicroorganism with one or more ORFs of interest, which microorganismcomprises one or more altered activities selected from the groupconsisting of omega hydroxyl fatty acid dehydrogenase activity, acyl-CoAoxidase activity, acyltransferase activity, thioesterase activity,monooxygenase activity and monooxygenase reductase activity.

In some embodiments, the nucleic acid reagent includes one or morerecombinase insertion sites. A recombinase insertion site is arecognition sequence on a nucleic acid molecule that participates in anintegration/recombination reaction by recombination proteins. Forexample, the recombination site for Cre recombinase is IoxP, which is a34 base pair sequence comprised of two 13 base pair inverted repeats(serving as the recombinase binding sites) flanking an 8 base pair coresequence (e.g., FIG. 1 of Sauer, B., Curr. Opin. Biotech. 5:521-527(1994)). Other examples of recombination sites include attB, attP, attL,and attR sequences, and mutants, fragments, variants and derivativesthereof, which are recognized by the recombination protein λ Int and bythe auxiliary proteins integration host factor (IHF), FIS andexcisionase (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557; 6,171,861;6,270,969; 6,277,608; and 6,720,140; U.S. patent application Ser. No.09/517,466, filed Mar. 2, 2000, and Ser. No. 09/732,914, filed Aug. 14,2003, and in U.S. patent publication no. 2002-0007051-A1; Landy, Curr.Opin. Biotech. 3:699-707 (1993)).

Examples of recombinase cloning nucleic acids are in Gateway® systems(Invitrogen, California), which include at least one recombination sitefor cloning a desired nucleic acid molecules in vivo or in vitro. Insome embodiments, the system utilizes vectors that contain at least twodifferent site-specific recombination sites, often based on thebacteriophage lambda system (e.g., att1 and att2), and are mutated fromthe wild-type (att0) sites. Each mutated site has a unique specificityfor its cognate partner att site (i.e., its binding partnerrecombination site) of the same type (for example attB1 with attP1, orattL1 with attR1) and will not cross-react with recombination sites ofthe other mutant type or with the wild-type att0 site. Different sitespecificities allow directional cloning or linkage of desired moleculesthus providing desired orientation of the cloned molecules. Nucleic acidfragments flanked by recombination sites are cloned and subcloned usingthe Gateway® system by replacing a selectable marker (for example, ccdB)flanked by att sites on the recipient plasmid molecule, sometimes termedthe Destination Vector. Desired clones are then selected bytransformation of a ccdB sensitive host strain and positive selectionfor a marker on the recipient molecule. Similar strategies for negativeselection (e.g., use of toxic genes) can be used in other organisms suchas thymidine kinase (TK) in mammals and insects.

A recombination system useful for engineering yeast is outlined briefly.The system makes use of the URA3 gene (e.g., for S. cerevisieae and C.albicans, for example) or URA4 and URA5 genes (e.g., for S. pombe, forexample) and toxicity of the nucleotide analogue 5-Fluoroorotic acid(5-FOA). The URA3 or URA4 and URA5 genes encode orotine-5′-monophosphate(OMP) dicarboxylase. Yeast with an active URA3 or URA4 and URA5 gene(phenotypically Ura+) convert 5-FOA to fluorodeoxyuridine, which istoxic to yeast cells. Yeast carrying a mutation in the appropriategene(s) or having a knock out of the appropriate gene(s) can grow in thepresence of 5-FOA, if the media is also supplemented with uracil.

A nucleic acid engineering construct can be made which may comprise theURA3 gene or cassette (for S. cerevisieae), flanked on either side bythe same nucleotide sequence in the same orientation. The URA3 cassettecomprises a promoter, the URA3 gene and a functional transcriptionterminator. Target sequences which direct the construct to a particularnucleic acid region of interest in the organism to be engineered areadded such that the target sequences are adjacent to and abut theflanking sequences on either side of the URA3 cassette. Yeast can betransformed with the engineering construct and plated on minimal mediawithout uracil. Colonies can be screened by PCR to determine thosetransformants that have the engineering construct inserted in the properlocation in the genome. Checking insertion location prior to selectingfor recombination of the ura3 cassette may reduce the number ofincorrect clones carried through to later stages of the procedure.Correctly inserted transformants can then be replica plated on minimalmedia containing 5-FOA to select for recombination of the URA3 cassetteout of the construct, leaving a disrupted gene and an identifiablefootprint (e.g., nucleic acid sequence) that can be use to verify thepresence of the disrupted gene. The technique described is useful fordisrupting or “knocking out” gene function, but also can be used toinsert genes or constructs into a host organisms genome in a targeted,sequence specific manner.

In certain embodiments, a nucleic acid reagent includes one or moretopoisomerase insertion sites. A topoisomerase insertion site is adefined nucleotide sequence recognized and bound by a site-specifictopoisomerase. For example, the nucleotide sequence 5′-(C/T)CCTT-3′ is atopoisomerase recognition site bound specifically by most poxvirustopoisomerases, including vaccinia virus DNA topoisomerase I. Afterbinding to the recognition sequence, the topoisomerase cleaves thestrand at the 3′-most thymidine of the recognition site to produce anucleotide sequence comprising 5′-(C/T)CCTT-PO4-TOPO, a complex of thetopoisomerase covalently bound to the 3′ phosphate via a tyrosine in thetopoisomerase (e.g., Shuman, J. Biol. Chem. 266:11372-11379, 1991;Sekiguchi and Shuman, Nucl. Acids Res. 22:5360-5365, 1994; U.S. Pat. No.5,766,891; PCT/US95/16099; and PCT/US98/12372). In comparison, thenucleotide sequence 5′-GCAACTT-3′ is a topoisomerase recognition sitefor type IA E. coli topoisomerase III. An element to be inserted oftenis combined with topoisomerase-reacted template and thereby incorporatedinto the nucleic acid reagent (e.g., World Wide Web URLinvitrogen.com/downloads/F-13512_Topo_Flyer.pdf; World Wide Web URLinvitrogen.com/content/sfs/brochures/710_021849%20_B_TOPOCloning_bro.pdf;TOPO TA Cloning® Kit and Zero Blunt® TOPO® Cloning Kit productinformation).

A nucleic acid reagent sometimes contains one or more origin ofreplication (ORI) elements. In some embodiments, a template comprisestwo or more ORIs, where one functions efficiently in one organism (e.g.,a bacterium) and another functions efficiently in another organism(e.g., a eukaryote, like yeast for example). In some embodiments, an ORImay function efficiently in one species (e.g., S. cerevisieae, forexample) and another ORI may function efficiently in a different species(e.g., S. pombe, for example). A nucleic acid reagent also sometimesincludes one or more transcription regulation sites.

A nucleic acid reagent can include one or more selection elements (e.g.,elements for selection of the presence of the nucleic acid reagent, andnot for activation of a promoter element which can be selectivelyregulated). Selection elements often are utilized using known processesto determine whether a nucleic acid reagent is included in a cell. Insome embodiments, a nucleic acid reagent includes two or more selectionelements, where one functions efficiently in one organism and anotherfunctions efficiently in another organism. Examples of selectionelements include, but are not limited to, (1) nucleic acid segments thatencode products that provide resistance against otherwise toxiccompounds (e.g., antibiotics); (2) nucleic acid segments that encodeproducts that are otherwise lacking in the recipient cell (e.g.,essential products, tRNA genes, auxotrophic markers); (3) nucleic acidsegments that encode products that suppress the activity of a geneproduct; (4) nucleic acid segments that encode products that can bereadily identified (e.g., phenotypic markers such as antibiotics (e.g.,β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellowfluorescent protein (YFP), red fluorescent protein (RFP), cyanfluorescent protein (CFP), and cell surface proteins); (5) nucleic acidsegments that bind products that are otherwise detrimental to cellsurvival and/or function; (6) nucleic acid segments that otherwiseinhibit the activity of any of the nucleic acid segments described inNos. 1-5 above (e.g., antisense oligonucleotides); (7) nucleic acidsegments that bind products that modify a substrate (e.g., restrictionendonucleases); (8) nucleic acid segments that can be used to isolate oridentify a desired molecule (e.g., specific protein binding sites); (9)nucleic acid segments that encode a specific nucleotide sequence thatcan be otherwise non-functional (e.g., for PCR amplification ofsubpopulations of molecules); (10) nucleic acid segments that, whenabsent, directly or indirectly confer resistance or sensitivity toparticular compounds; (11) nucleic acid segments that encode productsthat either are toxic or convert a relatively non-toxic compound to atoxic compound (e.g., Herpes simplex thymidine kinase, cytosinedeaminase) in recipient cells; (12) nucleic acid segments that inhibitreplication, partition or heritability of nucleic acid molecules thatcontain them; and/or (13) nucleic acid segments that encode conditionalreplication functions, e.g., replication in certain hosts or host cellstrains or under certain environmental conditions (e.g., temperature,nutritional conditions, and the like).

A nucleic acid reagent is of any form useful for in vivo transcriptionand/or translation. A nucleic acid sometimes is a plasmid, such as asupercoiled plasmid, sometimes is a yeast artificial chromosome (e.g.,YAC), sometimes is a linear nucleic acid (e.g., a linear nucleic acidproduced by PCR or by restriction digest), sometimes is single-strandedand sometimes is double-stranded. A nucleic acid reagent sometimes isprepared by an amplification process, such as a polymerase chainreaction (PCR) process or transcription-mediated amplification process(TMA). In TMA, two enzymes are used in an isothermal reaction to produceamplification products detected by light emission (see, e.g.,Biochemistry 1996 Jun. 25; 35(25):8429-38 and http addresswww.devicelink.com/ivdt/archive/00/11/007.html). Standard PCR processesare known (e.g., U.S. Pat. Nos. 4,683,202; 4,683,195; 4,965,188; and5,656,493), and generally are performed in cycles. Each cycle includesheat denaturation, in which hybrid nucleic acids dissociate; cooling, inwhich primer oligonucleotides hybridize; and extension of theoligonucleotides by a polymerase (i.e., Taq polymerase). An example of aPCR cyclical process is treating the sample at 95° C. for 5 minutes;repeating forty-five cycles of 95° C. for 1 minute, 59° C. for 1 minute,10 seconds, and 72° C. for 1 minute 30 seconds; and then treating thesample at 72° C. for 5 minutes. Multiple cycles frequently are performedusing a commercially available thermal cycler. PCR amplificationproducts sometimes are stored for a time at a lower temperature (e.g.,at 4° C.) and sometimes are frozen (e.g., at −20° C.) before analysis.

In some embodiments, a nucleic acid reagent, protein reagent, proteinfragment reagent or other reagent described herein is isolated orpurified. The term “isolated” as used herein refers to material removedfrom its original environment (e.g., the natural environment if it isnaturally occurring, or a host cell if expressed exogenously), and thusis altered “by the hand of man” from its original environment. The term“purified” as used herein with reference to molecules does not refer toabsolute purity. Rather, “purified” refers to a substance in acomposition that contains fewer substance species in the same class(e.g., nucleic acid or protein species) other than the substance ofinterest in comparison to the sample from which it originated.“Purified,” if a nucleic acid or protein for example, refers to asubstance in a composition that contains fewer nucleic acid species orprotein species other than the nucleic acid or protein of interest incomparison to the sample from which it originated. Sometimes, a proteinor nucleic acid is “substantially pure,” indicating that the protein ornucleic acid represents at least 50% of protein or nucleic acid on amass basis of the composition. Often, a substantially pure protein ornucleic acid is at least 75% on a mass basis of the composition, andsometimes at least 95% on a mass basis of the composition.

Engineering and Alteration Methods

Methods and compositions (e.g., nucleic acid reagents) described hereincan be used to generate engineered microorganisms. As noted above, theterm “engineered microorganism” as used herein refers to a modifiedorganism that includes one or more activities distinct from an activitypresent in a microorganism utilized as a starting point for modification(e.g., host microorganism or unmodified organism). Engineeredmicroorganisms typically arise as a result of a genetic modification,usually introduced or selected for, by one of skill in the art usingreadily available techniques. Non-limiting examples of methods usefulfor generating an altered activity include, introducing a heterologouspolynucleotide (e.g., nucleic acid or gene integration, also referred toas “knock in”), removing an endogenous polynucleotide, altering thesequence of an existing endogenous nucleic acid sequence (e.g.,site-directed mutagenesis), disruption of an existing endogenous nucleicacid sequence (e.g., knock outs and transposon or insertion elementmediated mutagenesis), selection for an altered activity where theselection causes a change in a naturally occurring activity that can bestably inherited (e.g., causes a change in a nucleic acid sequence inthe genome of the organism or in an epigenetic nucleic acid that isreplicated and passed on to daughter cells), PCR-based mutagenesis, andthe like. The term “mutagenesis” as used herein refers to anymodification to a nucleic acid (e.g., nucleic acid reagent, or hostchromosome, for example) that is subsequently used to generate a productin a host or modified organism. Non-limiting examples of mutagenesisinclude deletion, insertion, substitution, rearrangement, pointmutations, suppressor mutations and the like. Mutagenesis methods areknown in the art and are readily available to the artisan. Non-limitingexamples of mutagenesis methods are described herein and can also befound in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) MolecularCloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. Another non-limiting example of mutagenesis can beconducted using a Stratagene (San Diego, Calif.) “QuickChange” kitaccording to the manufacturer's instructions.

The term “genetic modification” as used herein refers to any suitablenucleic acid addition, removal or alteration that facilitates productionof a target fatty dicarboxylic acid product (e.g., sebacic ordodecanedioic acid) in an engineered microorganism. Geneticmodifications include, without limitation, insertion of one or morenucleotides in a native nucleic acid of a host organism in one or morelocations, deletion of one or more nucleotides in a native nucleic acidof a host organism in one or more locations, modification orsubstitution of one or more nucleotides in a native nucleic acid of ahost organism in one or more locations, insertion of a non-nativenucleic acid into a host organism (e.g., insertion of an autonomouslyreplicating vector), and removal of a non-native nucleic acid in a hostorganism (e.g., removal of a vector).

The term “heterologous polynucleotide” as used herein refers to anucleotide sequence not present in a host microorganism in someembodiments. In certain embodiments, a heterologous polynucleotide ispresent in a different amount (e.g., different copy number) than in ahost microorganism, which can be accomplished, for example, byintroducing more copies of a particular nucleotide sequence to a hostmicroorganism (e.g., the particular nucleotide sequence may be in anucleic acid autonomous of the host chromosome or may be inserted into achromosome). A heterologous polynucleotide is from a different organismin some embodiments, and in certain embodiments, is from the same typeof organism but from an outside source (e.g., a recombinant source).

In some embodiments, an organism engineered using the methods andnucleic acid reagents described herein can produce a fatty dicarboxylicacid (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid). In certain embodiments, an engineered microorganismdescribed herein that produces a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid)may comprise one ore more altered activities selected from the groupconsisting of omega oxo fatty acid dehydrogenase activity, omegahydroxyl fatty acid dehydrogenase activity, fatty acid synthaseactivity, acetyl CoA carboxylase activity, acyl-CoA oxidase activity,monooxygenase activity and monooxygenase reductase activity. In someembodiments, an engineered microorganism as described herein maycomprise a genetic modification that adds or increases the omega oxofatty acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, fatty acid synthase activity, acetyl CoAcarboxylase activity, acyl-CoA oxidase activity, monooxygenase activityand monooxygenase reductase activity.

In certain embodiments, an engineered microorganism described herein cancomprise an altered thioesterase activity. In some embodiments, theengineered microorganism may comprise a genetic alteration that adds orincreases a thioesterase activity. In some embodiments, the engineeredmicroorganism comprising a genetic alteration that adds or increases athioesterase activity, may further comprise a heterologouspolynucleotide encoding a polypeptide having thioesterase activity.

The term “altered activity” as used herein refers to an activity in anengineered microorganism that is added or modified relative to the hostmicroorganism (e.g., added, increased, reduced, inhibited or removedactivity). An activity can be altered by introducing a geneticmodification to a host microorganism that yields an engineeredmicroorganism having added, increased, reduced, inhibited or removedactivity.

An added activity often is an activity not detectable in a hostmicroorganism. An increased activity generally is an activity detectablein a host microorganism that has been increased in an engineeredmicroorganism. An activity can be increased to any suitable level forproduction of a target fatty dicarboxylic acid product (e.g., sebacic ordodecanedioic acid), including but not limited to less than 2-fold(e.g., about 10% increase to about 99% increase; about 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% increase), 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, of 10-fold increase, or greater thanabout 10-fold increase. A reduced or inhibited activity generally is anactivity detectable in a host microorganism that has been reduced orinhibited in an engineered microorganism. An activity can be reduced toundetectable levels in some embodiments, or detectable levels in certainembodiments. An activity can be decreased to any suitable level forproduction of a target fatty dicarboxylic acid product (e.g., sebacic ordodecanedioic acid), including but not limited to less than 2-fold(e.g., about 10% decrease to about 99% decrease; about 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% decrease), 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, of 10-fold decrease, or greater thanabout 10-fold decrease.

An altered activity sometimes is an activity not detectable in a hostorganism and is added to an engineered organism. An altered activityalso may be an activity detectable in a host organism and is increasedin an engineered organism. An activity may be added or increased byincreasing the number of copies of a polynucleotide that encodes apolypeptide having a target activity, in some embodiments. In someembodiments, the activity of a native polypeptide can be increased byincreasing in the modified organism the number of copies of apolynucleotide that encodes the polypeptide (e.g., introducing 1 toabout 100 additional copies of the polynucleotide (e.g., introducing 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22,24, 26, 28, 30 or more additional copies of the polynucleotide). Incertain embodiments an activity can be added or increased by insertinginto a host microorganism a polynucleotide that encodes a heterologouspolypeptide having the added activity or encodes a modified endogenouspolypeptide. In such embodiments, 1 to about 100 copies of thepolynucleotide can be introduced (e.g., introducing 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30copies). A “modified endogenous polypeptide” often has an activitydifferent than an activity of a native polypeptide counterpart (e.g.,different catalytic activity and/or different substrate specificity),and often is active (e.g., an activity (e.g., substrate turnover) isdetectable). In certain embodiments, an activity can be added orincreased by inserting into a host microorganism a heterologouspolynucleotide that is (i) operably linked to another polynucleotidethat encodes a polypeptide having the added activity, and (ii) upregulates production of the polynucleotide. Thus, an activity can beadded or increased by inserting or modifying a regulatory polynucleotideoperably linked to another polynucleotide that encodes a polypeptidehaving the target activity. In certain embodiments, an activity can beadded or increased by subjecting a host microorganism to a selectiveenvironment and screening for microorganisms that have a detectablelevel of the target activity. Examples of a selective environmentinclude, without limitation, a medium containing a substrate that a hostorganism can process and a medium lacking a substrate that a hostorganism can process.

An altered activity sometimes is an activity detectable in a hostorganism and is reduced, inhibited or removed (i.e., not detectable) inan engineered organism. An activity may be reduced or removed bydecreasing the number of copies of a polynucleotide that encodes apolypeptide having a target activity, in some embodiments. In someembodiments, an activity can be reduced or removed by (i) inserting apolynucleotide within a polynucleotide that encodes a polypeptide havingthe target activity (disruptive insertion), and/or (ii) removing aportion of or all of a polynucleotide that encodes a polypeptide havingthe target activity (deletion or knock out, respectively). In certainembodiments, an activity can be reduced or removed by inserting into ahost microorganism a heterologous polynucleotide that is (i) operablylinked to another polynucleotide that encodes a polypeptide having thetarget activity, and (ii) down regulates production of thepolynucleotide. Thus, an activity can be reduced or removed by insertingor modifying a regulatory polynucleotide operably linked to anotherpolynucleotide that encodes a polypeptide having the target activity.

An activity also can be reduced or removed by (i) inhibiting apolynucleotide that encodes a polypeptide having the activity or (ii)inhibiting a polynucleotide operably linked to another polynucleotidethat encodes a polypeptide having the activity. A polynucleotide can beinhibited by a suitable technique known in the art, such as bycontacting an RNA encoded by the polynucleotide with a specificinhibitory RNA (e.g., RNAi, siRNA, ribozyme). An activity also can bereduced or removed by contacting a polypeptide having the activity witha molecule that specifically inhibits the activity (e.g., enzymeinhibitor, antibody). In certain embodiments, an activity can be reducedor removed by subjecting a host microorganism to a selective environmentand screening for microorganisms that have a reduced level or removal ofthe target activity.

In some embodiments, an untranslated ribonucleic acid, or a cDNA can beused to reduce the expression of a particular activity or enzyme. Forexample, a microorganism can be engineered by genetic modification toexpress a nucleic acid reagent that reduces the expression of anactivity by producing an RNA molecule that is partially or substantiallyhomologous to a nucleic acid sequence of interest which encodes theactivity of interest. The RNA molecule can bind to the nucleic acidsequence of interest and inhibit the nucleic acid sequence fromperforming its natural function, in certain embodiments. In someembodiments, the RNA may alter the nucleic acid sequence of interestwhich encodes the activity of interest in a manner that the nucleic acidsequence of interest is no longer capable of performing its naturalfunction (e.g., the action of a ribozyme for example).

In certain embodiments, nucleotide sequences sometimes are added to,modified or removed from one or more of the nucleic acid reagentelements, such as the promoter, 5′UTR, target sequence, or 3′UTRelements, to enhance, potentially enhance, reduce, or potentially reducetranscription and/or translation before or after such elements areincorporated in a nucleic acid reagent. In some embodiments, one or moreof the following sequences may be modified or removed if they arepresent in a 5′UTR: a sequence that forms a stable secondary structure(e.g., quadruplex structure or stem loop stem structure (e.g., EMBLsequences X12949, AF274954, AF139980, AF152961, S95936, U194144,AF116649 or substantially identical sequences that form such stem loopstem structures)); a translation initiation codon upstream of the targetnucleotide sequence start codon; a stop codon upstream of the targetnucleotide sequence translation initiation codon; an ORF upstream of thetarget nucleotide sequence translation initiation codon; an ironresponsive element (IRE) or like sequence; and a 5′ terminaloligopyrimidine tract (TOP, e.g., consisting of 5-15 pyrimidinesadjacent to the cap). A translational enhancer sequence and/or aninternal ribosome entry site (IRES) sometimes is inserted into a 5′UTR(e.g., EMBL nucleotide sequences J04513, X87949, M95825, M12783,AF025841, AF013263, AF006822, M17169, M13440, M22427, D14838 and M17446and substantially identical nucleotide sequences).

An AU-rich element (ARE, e.g., AUUUA repeats) and/or splicing junctionthat follows a non-sense codon sometimes is removed from or modified ina 3′UTR. A polyadenosine tail sometimes is inserted into a 3′UTR if noneis present, sometimes is removed if it is present, and adenosinemoieties sometimes are added to or removed from a polyadenosine tailpresent in a 3′UTR. Thus, some embodiments are directed to a processcomprising: determining whether any nucleotide sequences that increase,potentially increase, reduce or potentially reduce translationefficiency are present in the elements, and adding, removing ormodifying one or more of such sequences if they are identified. Certainembodiments are directed to a process comprising: determining whetherany nucleotide sequences that increase or potentially increasetranslation efficiency are not present in the elements, andincorporating such sequences into the nucleic acid reagent.

In some embodiments, an activity can be altered by modifying thenucleotide sequence of an ORF. An ORF sometimes is mutated or modified(for example, by point mutation, deletion mutation, insertion mutation,PCR based mutagenesis and the like) to alter, enhance or increase,reduce, substantially reduce or eliminate the activity of the encodedprotein or peptide. The protein or peptide encoded by a modified ORFsometimes is produced in a lower amount or may not be produced atdetectable levels, and in other embodiments, the product or proteinencoded by the modified ORF is produced at a higher level (e.g., codonssometimes are modified so they are compatible with tRNA's preferentiallyused in the host organism or engineered organism). To determine therelative activity, the activity from the product of the mutated ORF (orcell containing it) can be compared to the activity of the product orprotein encoded by the unmodified ORF (or cell containing it).

In some embodiments, an ORF nucleotide sequence sometimes is mutated ormodified to alter the triplet nucleotide sequences used to encode aminoacids (e.g., amino acid codon triplets, for example). Modification ofthe nucleotide sequence of an ORF to alter codon triplets sometimes isused to change the codon found in the original sequence to better matchthe preferred codon usage of the organism in which the ORF or nucleicacid reagent will be expressed. The codon usage, and therefore the codontriplets encoded by a nucleic acid sequence, in bacteria may bedifferent from the preferred codon usage in eukaryotes, like yeast orplants for example. Preferred codon usage also may be different betweenbacterial species. In certain embodiments an ORF nucleotide sequencessometimes is modified to eliminate codon pairs and/or eliminate mRNAsecondary structures that can cause pauses during translation of themRNA encoded by the ORF nucleotide sequence. Translational pausingsometimes occurs when nucleic acid secondary structures exist in anmRNA, and sometimes occurs due to the presence of codon pairs that slowthe rate of translation by causing ribosomes to pause. In someembodiments, the use of lower abundance codon triplets can reducetranslational pausing due to a decrease in the pause time needed to loada charged tRNA into the ribosome translation machinery. Therefore, toincrease transcriptional and translational efficiency in bacteria (e.g.,where transcription and translation are concurrent, for example) or toincrease translational efficiency in eukaryotes (e.g., wheretranscription and translation are functionally separated), thenucleotide sequence of a nucleotide sequence of interest can be alteredto better suit the transcription and/or translational machinery of thehost and/or genetically modified microorganism. In certain embodiments,slowing the rate of translation by the use of lower abundance codons,which slow or pause the ribosome, can lead to higher yields of thedesired product due to an increase in correctly folded proteins and areduction in the formation of inclusion bodies.

Codons can be altered and optimized according to the preferred usage bya given organism by determining the codon distribution of the nucleotidesequence donor organism and comparing the distribution of codons to thedistribution of codons in the recipient or host organism. Techniquesdescribed herein (e.g., site directed mutagenesis and the like) can thenbe used to alter the codons accordingly. Comparisons of codon usage canbe done by hand or using nucleic acid analysis software commerciallyavailable to the artisan.

Modification of the nucleotide sequence of an ORF also can be used tocorrect codon triplet sequences that have diverged in differentorganisms. For example, certain yeast (e.g., C. tropicalis and C.maltosa) use the amino acid triplet CUG (e.g., CTG in the DNA sequence)to encode serine. CUG typically encodes leucine in most organisms. Inorder to maintain the correct amino acid in the resultant polypeptide orprotein, the CUG codon must be altered to reflect the organism in whichthe nucleic acid reagent will be expressed. Thus, if an ORF from abacterial donor is to be expressed in either Candida yeast strainmentioned above, the heterologous nucleotide sequence must first bealtered or modified to the appropriate leucine codon. Therefore, in someembodiments, the nucleotide sequence of an ORF sometimes is altered ormodified to correct for differences that have occurred in the evolutionof the amino acid codon triplets between different organisms. In someembodiments, the nucleotide sequence can be left unchanged at aparticular amino acid codon, if the amino acid encoded is a conservativeor neutral change in amino acid when compared to the originally encodedamino acid.

In some embodiments, an activity can be altered by modifyingtranslational regulation signals, like a stop codon for example. A stopcodon at the end of an ORF sometimes is modified to another stop codon,such as an amber stop codon described above. In some embodiments, a stopcodon is introduced within an ORF, sometimes by insertion or mutation ofan existing codon. An ORF comprising a modified terminal stop codonand/or internal stop codon often is translated in a system comprising asuppressor tRNA that recognizes the stop codon. An ORF comprising a stopcodon sometimes is translated in a system comprising a suppressor tRNAthat incorporates an unnatural amino acid during translation of thetarget protein or target peptide. Methods for incorporating unnaturalamino acids into a target protein or peptide are known, which include,for example, processes utilizing a heterologous tRNA/synthetase pair,where the tRNA recognizes an amber stop codon and is loaded with anunnatural amino acid (e.g., World Wide Web URLiupac.org/news/prize/2003/wang.pdf).

Depending on the portion of a nucleic acid reagent (e.g., Promoter, 5′or 3′ UTR, ORI, ORF, and the like) chosen for alteration (e.g., bymutagenesis, introduction or deletion, for example) the modificationsdescribed above can alter a given activity by (i) increasing ordecreasing feedback inhibition mechanisms, (ii) increasing or decreasingpromoter initiation, (iii) increasing or decreasing translationinitiation, (iv) increasing or decreasing translational efficiency, (v)modifying localization of peptides or products expressed from nucleicacid reagents described herein, or (vi) increasing or decreasing thecopy number of a nucleotide sequence of interest, (vii) expression of ananti-sense RNA, RNAi, siRNA, ribozyme and the like. In some embodiments,alteration of a nucleic acid reagent or nucleotide sequence can alter aregion involved in feedback inhibition (e.g., 5′ UTR, promoter and thelike). A modification sometimes is made that can add or enhance bindingof a feedback regulator and sometimes a modification is made that canreduce, inhibit or eliminate binding of a feedback regulator.

In certain embodiments, alteration of a nucleic acid reagent ornucleotide sequence can alter sequences involved in transcriptioninitiation (e.g., promoters, 5′ UTR, and the like). A modificationsometimes can be made that can enhance or increase initiation from anendogenous or heterologous promoter element. A modification sometimescan be made that removes or disrupts sequences that increase or enhancetranscription initiation, resulting in a decrease or elimination oftranscription from an endogenous or heterologous promoter element.

In some embodiments, alteration of a nucleic acid reagent or nucleotidesequence can alter sequences involved in translational initiation ortranslational efficiency (e.g., 5′ UTR, 3′ UTR, codon triplets of higheror lower abundance, translational terminator sequences and the like, forexample). A modification sometimes can be made that can increase ordecrease translational initiation, modifying a ribosome binding site forexample. A modification sometimes can be made that can increase ordecrease translational efficiency. Removing or adding sequences thatform hairpins and changing codon triplets to a more or less preferredcodon are non-limiting examples of genetic modifications that can bemade to alter translation initiation and translation efficiency.

In certain embodiments, alteration of a nucleic acid reagent ornucleotide sequence can alter sequences involved in localization ofpeptides, proteins or other desired products (e.g., a sebacic acid ordodecanedioic acid, for example). A modification sometimes can be madethat can alter, add or remove sequences responsible for targeting apolypeptide, protein or product to an intracellular organelle, theperiplasm, cellular membranes, or extracellularly. Transport of aheterologous product to a different intracellular space orextracellularly sometimes can reduce or eliminate the formation ofinclusion bodies (e.g., insoluble aggregates of the desired product).

In some embodiments, alteration of a nucleic acid reagent or nucleotidesequence can alter sequences involved in increasing or decreasing thecopy number of a nucleotide sequence of interest. A modificationsometimes can be made that increases or decreases the number of copiesof an ORF stably integrated into the genome of an organism or on anepigenetic nucleic acid reagent. Non-limiting examples of alterationsthat can increase the number of copies of a sequence of interestinclude, adding copies of the sequence of interest by duplication ofregions in the genome (e.g., adding additional copies by recombinationor by causing gene amplification of the host genome, for example),cloning additional copies of a sequence onto a nucleic acid reagent, oraltering an ORI to increase the number of copies of an epigeneticnucleic acid reagent. Non-limiting examples of alterations that candecrease the number of copies of a sequence of interest include,removing copies of the sequence of interest by deletion or disruption ofregions in the genome, removing additional copies of the sequence fromepigenetic nucleic acid reagents, or altering an ORI to decrease thenumber of copies of an epigenetic nucleic acid reagent.

In certain embodiments, increasing or decreasing the expression of anucleotide sequence of interest can also be accomplished by altering,adding or removing sequences involved in the expression of an anti-senseRNA, RNAi, siRNA, ribozyme and the like. The methods described above canbe used to modify expression of anti-sense RNA, RNAi, siRNA, ribozymeand the like.

The methods and nucleic acid reagents described herein can be used togenerate genetically modified microorganisms with altered activities incellular processes involved in a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid)synthesis. In some embodiments, an engineered microorganism describedherein may comprise an increased number of copies of an endogenouspolynucleotide encoding a polypeptide having omega oxo fatty aciddehydrogenase activity. In certain embodiments, an engineeredmicroorganism described herein may comprise an increased number ofcopies of an endogenous polynucleotide encoding a polypeptide havingomega hydroxyl fatty acid dehydrogenase activity. In some embodiments,an engineered microorganism described herein may comprise a heterologouspolynucleotide encoding a polypeptide having omega oxo fatty aciddehydrogenase activity. In some embodiments, an engineered microorganismdescribed herein may comprise a heterologous polynucleotide encoding apolypeptide having omega hydroxyl fatty acid dehydrogenase activity. Insome embodiments, the heterologous polynucleotide can be from abacterium. In some embodiments, the bacterium can be an Acinetobacter,Nocardia, Pseudomonas or Xanthobacter bacterium.

In some embodiments, an engineered microorganism described herein maycomprise a heterologous polynucleotide encoding a polypeptide havingmonooxygenase activity. In certain embodiments, the heterologouspolynucleotide can be from a bacterium. In some embodiments, thebacterium can be a Bacillus bacterium. In certain embodiments, theBacillus bacterium is B. megaterium.

In certain embodiments, an engineered microorganism described herein maycomprise a genetic modification that reduces omega hydroxyl fatty acidconversion. In some embodiments, the genetic modification can reduceomega hydroxyl fatty acid dehydrogenase activity. In certainembodiments, an engineered microorganism described herein may comprise agenetic modification that reduces beta-oxidation activity. In someembodiments, the genetic modification can reduce a target activitydescribed herein.

Engineered microorganisms that produce a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid),as described herein, can comprise an altered monooxygenase activity, incertain embodiments. In some embodiments, the engineered microorganismdescribed herein may comprise a genetic modification that alters themonooxygenase activity. In certain embodiments, the engineeredmicroorganism described herein can comprise an increase number of copiesof an endogenous polynucleotide encoding a polypeptide havingmonooxygenase activity. In some embodiments, the engineeredmicroorganism described herein can comprise a heterologouspolynucleotide encoding a polypeptide having monooxygenase activity. Incertain embodiments, the heterologous polynucleotide can be from abacterium. In some embodiments, the bacterium can be a Bacillusbacterium. In certain embodiments, the Bacillus bacterium is B.megaterium. In some embodiments, the genetic modification can reduce apolyketide synthase activity.

Engineered microorganisms that produce a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid),as described herein, can comprise an altered thioesterase activity, incertain embodiments. In some embodiments, the engineered microorganismmay comprise a genetic modification that adds or increases thethioesterase activity. In certain embodiments, the engineeredmicroorganism may comprise a heterologous polynucleotide encoding apolypeptide having thioesterase activity.

In some embodiments, the engineered microorganism with an alteredthioesterase activity may comprise an altered omega oxo fatty aciddehydrogenase activity. In certain embodiments, the engineeredmicroorganism with an altered thioesterase activity may comprise agenetic modification that adds or increases omega oxo fatty aciddehydrogenase activity. In some embodiments, the engineeredmicroorganism may comprise a heterologous polynucleotide encoding apolypeptide having altered omega oxo fatty acid dehydrogenase activity.In certain embodiments, the heterologous polynucleotide can be from abacterium. In some embodiments, the bacterium can be an Acinetobacter,Nocardia, Pseudomonas or Xanthobacter bacterium.

Engineered microorganisms that produce a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid),as described herein, can comprise an altered omega hydroxyl fatty aciddehydrogenase activity. In certain embodiments, the engineeredmicroorganism may comprise a genetic modification that adds or increasesthe omega hydroxyl fatty acid dehydrogenase activity. In certainembodiments, the engineered microorganism may comprise a heterologouspolynucleotide encoding a polypeptide having altered omega hydroxylfatty acid dehydrogenase activity. In some embodiments, the heterologouspolynucleotide is from a bacterium. In certain embodiments, thebacterium can be an Acinetobacter, Nocardia, Pseudomonas or Xanthobacterbacterium. In some embodiments, the engineered microorganism can be aeukaryote. In certain embodiments, the eukaryote can be a yeast. In someembodiments, the eukaryote may be a fungus. In certain embodiments, theyeast can be a Candida yeast. In some embodiments, the Candida yeast maybe C. troplicalis. In certain embodiments, the fungus can be a Yarrowiafungus. In some embodiments the Yarrowia fungus may be Y. lipolytica. Incertain embodiments, the fungus can be an Aspergillus fungus. In someembodiments, the Aspergillus fungus may be A. parasiticus or A.nidulans. In some embodiments, an engineered microorganism as describedabove may comprise a genetic modification that reduces omega hydroxylfatty acid conversion. In certain embodiments, the genetic modificationcan reduce omega hydroxyl fatty acid dehydrogenase activity. In someembodiments the genetic may reduce beta-oxidation activity. In certainembodiments, the genetic modification may reduce a target activitydescribed herein.

Engineered microorganisms can be prepared by altering, introducing orremoving nucleotide sequences in the host genome or in stably maintainedepigenetic nucleic acid reagents, as noted above. The nucleic acidreagents use to alter, introduce or remove nucleotide sequences in thehost genome or epigenetic nucleic acids can be prepared using themethods described herein or available to the artisan.

Nucleic acid sequences having a desired activity can be isolated fromcells of a suitable organism using lysis and nucleic acid purificationprocedures described in a known reference manual (e.g., Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning: a LaboratoryManual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) orusing commercially available cell lysis and DNA purification reagentsand kits. In some embodiments, nucleic acids used to engineermicroorganisms can be provided for conducting methods described hereinafter processing of the organism containing the nucleic acid. Forexample, the nucleic acid of interest may be extracted, isolated,purified or amplified from a sample (e.g., from an organism of interestor culture containing a plurality of organisms of interest, like yeastor bacteria for example). The term “isolated” as used herein refers tonucleic acid removed from its original environment (e.g., the naturalenvironment if it is naturally occurring, or a host cell if expressedexogenously), and thus is altered “by the hand of man” from its originalenvironment. An isolated nucleic acid generally is provided with fewernon-nucleic acid components (e.g., protein, lipid) than the amount ofcomponents present in a source sample. A composition comprising isolatedsample nucleic acid can be substantially isolated (e.g., about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% free ofnon-nucleic acid components). The term “purified” as used herein refersto sample nucleic acid provided that contains fewer nucleic acid speciesthan in the sample source from which the sample nucleic acid is derived.A composition comprising sample nucleic acid may be substantiallypurified (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or greater than 99% free of other nucleic acid species). The term“amplified” as used herein refers to subjecting nucleic acid of a cell,organism or sample to a process that linearly or exponentially generatesamplicon nucleic acids having the same or substantially the samenucleotide sequence as the nucleotide sequence of the nucleic acid inthe sample, or portion thereof. As noted above, the nucleic acids usedto prepare nucleic acid reagents as described herein can be subjected tofragmentation or cleavage.

Amplification of nucleic acids is sometimes necessary when dealing withorganisms that are difficult to culture. Where amplification may bedesired, any suitable amplification technique can be utilized.Non-limiting examples of methods for amplification of polynucleotidesinclude, polymerase chain reaction (PCR); ligation amplification (orligase chain reaction (LCR)); amplification methods based on the use ofQ-beta replicase or template-dependent polymerase (see US PatentPublication Number US20050287592); helicase-dependant isothermalamplification (Vincent et al., “Helicase-dependent isothermal DNAamplification”. EMBO reports 5 (8): 795-800 (2004)); strand displacementamplification (SDA); thermophilic SDA nucleic acid sequence basedamplification (3SR or NASBA) and transcription-associated amplification(TAA). Non-limiting examples of PCR amplification methods includestandard PCR, AFLP-PCR, Allele-specific PCR, Alu-PCR, Asymmetric PCR,Colony PCR, Hot start PCR, Inverse PCR (I PCR), In situ PCR (ISH),Intersequence-specific PCR (ISSR-PCR), Long PCR, Multiplex PCR, NestedPCR, Quantitative PCR, Reverse Transcriptase PCR (RT-PCR), Real TimePCR, Single cell PCR, Solid phase PCR, combinations thereof, and thelike. Reagents and hardware for conducting PCR are commerciallyavailable.

Protocols for conducting the various type of PCR listed above arereadily available to the artisan. PCR conditions can be dependent uponprimer sequences, target abundance, and the desired amount ofamplification, and therefore, one of skill in the art may choose from anumber of PCR protocols available (see, e.g., U.S. Pat. Nos. 4,683,195and 4,683,202; and PCR Protocols: A Guide to Methods and Applications,Innis et al., eds, 1990. PCR often is carried out as an automatedprocess with a thermostable enzyme. In this process, the temperature ofthe reaction mixture is cycled through a denaturing region, aprimer-annealing region, and an extension reaction region automatically.Machines specifically adapted for this purpose are commerciallyavailable. A non-limiting example of a PCR protocol that may be suitablefor embodiments described herein is, treating the sample at 95° C. for 5minutes; repeating forty-five cycles of 95° C. for 1 minute, 59° C. for1 minute, 10 seconds, and 72° C. for 1 minute 30 seconds; and thentreating the sample at 72° C. for 5 minutes. Additional PCR protocolsare described in the example section. Multiple cycles frequently areperformed using a commercially available thermal cycler. Suitableisothermal amplification processes known and selected by the person ofordinary skill in the art also may be applied, in certain embodiments.In some embodiments, nucleic acids encoding polypeptides with a desiredactivity can be isolated by amplifying the desired sequence from anorganism having the desired activity using oligonucleotides or primersdesigned based on sequences described herein.

Amplified, isolated and/or purified nucleic acids can be cloned into therecombinant DNA vectors described in Figures herein or into suitablecommercially available recombinant DNA vectors. Cloning of nucleic acidsequences of interest into recombinant DNA vectors can facilitatefurther manipulations of the nucleic acids for preparation of nucleicacid reagents, (e.g., alteration of nucleotide sequences by mutagenesis,homologous recombination, amplification and the like, for example).Standard cloning procedures (e.g., enzymic digestion, ligation, and thelike) are known (e.g., described in Maniatis, T., E. F. Fritsch and J.Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

In some embodiments, nucleic acid sequences prepared by isolation oramplification can be used, without any further modification, to add anactivity to a microorganism and thereby create a genetically modified orengineered microorganism. In certain embodiments, nucleic acid sequencesprepared by isolation or amplification can be genetically modified toalter (e.g., increase or decrease, for example) a desired activity. Insome embodiments, nucleic acids, used to add an activity to an organism,sometimes are genetically modified to optimize the heterologouspolynucleotide sequence encoding the desired activity (e.g., polypeptideor protein, for example). The term “optimize” as used herein can referto alteration to increase or enhance expression by preferred codonusage. The term optimize can also refer to modifications to the aminoacid sequence to increase the activity of a polypeptide or protein, suchthat the activity exhibits a higher catalytic activity as compared tothe “natural” version of the polypeptide or protein.

Nucleic acid sequences of interest can be genetically modified usingmethods known in the art. Mutagenesis techniques are particularly usefulfor small scale (e.g., 1, 2, 5, 10 or more nucleotides) or large scale(e.g., 50, 100, 150, 200, 500, or more nucleotides) geneticmodification. Mutagenesis allows the artisan to alter the geneticinformation of an organism in a stable manner, either naturally (e.g.,isolation using selection and screening) or experimentally by the use ofchemicals, radiation or inaccurate DNA replication (e.g., PCRmutagenesis). In some embodiments, genetic modification can be performedby whole scale synthetic synthesis of nucleic acids, using a nativenucleotide sequence as the reference sequence, and modifying nucleotidesthat can result in the desired alteration of activity. Mutagenesismethods sometimes are specific or targeted to specific regions ornucleotides (e.g., site-directed mutagenesis, PCR-based site-directedmutagenesis, and in vitro mutagenesis techniques such as transplacementand in vivo oligonucleotide site-directed mutagenesis, for example).Mutagenesis methods sometimes are non-specific or random with respect tothe placement of genetic modifications (e.g., chemical mutagenesis,insertion element (e.g., insertion or transposon elements) andinaccurate PCR based methods, for example).

Site directed mutagenesis is a procedure in which a specific nucleotideor specific nucleotides in a DNA molecule are mutated or altered. Sitedirected mutagenesis typically is performed using a nucleic acidsequence of interest cloned into a circular plasmid vector.Site-directed mutagenesis requires that the wild type sequence be knownand used a platform for the genetic alteration. Site-directedmutagenesis sometimes is referred to as oligonucleotide-directedmutagenesis because the technique can be performed usingoligonucleotides which have the desired genetic modificationincorporated into the complement a nucleotide sequence of interest. Thewild type sequence and the altered nucleotide are allowed to hybridizeand the hybridized nucleic acids are extended and replicated using a DNApolymerase. The double stranded nucleic acids are introduced into a host(e.g., E. coli, for example) and further rounds of replication arecarried out in vivo. The transformed cells carrying the mutated nucleicacid sequence are then selected and/or screened for those cells carryingthe correctly mutagenized sequence. Cassette mutagenesis and PCR-basedsite-directed mutagenesis are further modifications of the site-directedmutagenesis technique. Site-directed mutagenesis can also be performedin vivo (e.g., transplacement “pop-in pop-out”, In vivo site-directedmutagenesis with synthetic oligonucleotides and the like, for example).

PCR-based mutagenesis can be performed using PCR with oligonucleotideprimers that contain the desired mutation or mutations. The techniquefunctions in a manner similar to standard site-directed mutagenesis,with the exception that a thermocycler and PCR conditions are used toreplace replication and selection of the clones in a microorganism host.As PCR-based mutagenesis also uses a circular plasmid vector, theamplified fragment (e.g., linear nucleic acid molecule) containing theincorporated genetic modifications can be separated from the plasmidcontaining the template sequence after a sufficient number of rounds ofthermocycler amplification, using standard electrophorectic procedures.A modification of this method uses linear amplification methods and apair of mutagenic primers that amplify the entire plasmid. The proceduretakes advantage of the E. coli Dam methylase system which causes DNAreplicated in vivo to be sensitive to the restriction endonucleasesDpnI. PCR synthesized DNA is not methylated and is therefore resistantto DpnI. This approach allows the template plasmid to be digested,leaving the genetically modified, PCR synthesized plasmids to beisolated and transformed into a host bacteria for DNA repair andreplication, thereby facilitating subsequent cloning and identificationsteps. A certain amount of randomness can be added to PCR-based siteddirected mutagenesis by using partially degenerate primers.

Recombination sometimes can be used as a tool for mutagenesis.Homologous recombination allows the artisan to specifically targetregions of known sequence for insertion of heterologous nucleotidesequences using the host organisms natural DNA replication and repairenzymes. Homologous recombination methods sometimes are referred to as“pop in pop out” mutagenesis, transplacement, knock out mutagenesis orknock in mutagenesis. Integration of a nucleic acid sequence into a hostgenome is a single cross over event, which inserts the entire nucleicacid reagent (e.g., pop in). A second cross over event excises all but aportion of the nucleic acid reagent, leaving behind a heterologoussequence, often referred to as a “footprint” (e.g., pop out).Mutagenesis by insertion (e.g., knock in) or by double recombinationleaving behind a disrupting heterologous nucleic acid (e.g., knock out)both server to disrupt or “knock out” the function of the gene ornucleic acid sequence in which insertion occurs. By combining selectablemarkers and/or auxotrophic markers with nucleic acid reagents designedto provide the appropriate nucleic acid target sequences, the artisancan target a selectable nucleic acid reagent to a specific region, andthen select for recombination events that “pop out” a portion of theinserted (e.g., “pop in”) nucleic acid reagent.

Such methods take advantage of nucleic acid reagents that have beenspecifically designed with known target nucleic acid sequences at ornear a nucleic acid or genomic region of interest. Popping out typicallyleaves a “foot print” of left over sequences that remain after therecombination event. The left over sequence can disrupt a gene andthereby reduce or eliminate expression of that gene. In someembodiments, the method can be used to insert sequences, upstream ordownstream of genes that can result in an enhancement or reduction inexpression of the gene. In certain embodiments, new genes can beintroduced into the genome of a host organism using similarrecombination or “pop in” methods. An example of a yeast recombinationsystem using the ura3 gene and 5-FOA were described briefly above andfurther detail is presented herein.

A method for modification is described in Alani et al., “A method forgene disruption that allows repeated use of URA3 selection in theconstruction of multiply disrupted yeast strains”, Genetics116(4):541-545 August 1987. The original method uses a Ura3 cassettewith 1000 base pairs (bp) of the same nucleotide sequence cloned in thesame orientation on either side of the URA3 cassette. Targetingsequences of about 50 bp are added to each side of the construct. Thedouble stranded targeting sequences are complementary to sequences inthe genome of the host organism. The targeting sequences allowsite-specific recombination in a region of interest. The modification ofthe original technique replaces the two 1000 bp sequence direct repeatswith two 200 bp direct repeats. The modified method also uses 50 bptargeting sequences. The modification reduces or eliminatesrecombination of a second knock out into the 1000 bp repeat left behindin a first mutagenesis, therefore allowing multiply knocked out yeast.Additionally, the 200 bp sequences used herein are uniquely designed,self-assembling sequences that leave behind identifiable footprints. Thetechnique used to design the sequences incorporate design features suchas low identity to the yeast genome, and low identity to each other.Therefore a library of the self-assembling sequences can be generated toallow multiple knockouts in the same organism, while reducing oreliminating the potential for integration into a previous knockout.

As noted above, the URA3 cassette makes use of the toxicity of 5-FOA inyeast carrying a functional URA3 gene. Uracil synthesis deficient yeastare transformed with the modified URA3 cassette, using standard yeasttransformation protocols, and the transformed cells are plated onminimal media minus uracil. In some embodiments, PCR can be used toverify correct insertion into the region of interest in the host genome,and certain embodiments the PCR step can be omitted. Inclusion of thePCR step can reduce the number of transformants that need to be counterselected to “pop out” the URA3 cassette. The transformants (e.g., all orthe ones determined to be correct by PCR, for example) can then becounter-selected on media containing 5-FOA, which will select forrecombination out (e.g., popping out) of the URA3 cassette, thusrendering the yeast ura3 deficient again, and resistant to 5-FOAtoxicity. Targeting sequences used to direct recombination events tospecific regions are presented herein. A modification of the methoddescribed above can be used to integrate genes in to the chromosome,where after recombination a functional gene is left in the chromosomenext to the 200 bp footprint.

In some embodiments, other auxotrophic or dominant selection markers canbe used in place of URA3 (e.g., an auxotrophic selectable marker), withthe appropriate change in selection media and selection agents.Auxotrophic selectable markers are used in strains deficient forsynthesis of a required biological molecule (e.g., amino acid ornucleoside, for example). Non-limiting examples of additionalauxotrophic markers include; HIS3, TRP1, LEU2, LEU2-d, and LYS2. Certainauxotrophic markers (e.g., URA3 and LYS2) allow counter selection toselect for the second recombination event that pops out all but one ofthe direct repeats of the recombination construct. HIS3 encodes anactivity involved in histidine synthesis. TRP1 encodes an activityinvolved in tryptophan synthesis. LEU2 encodes an activity involved inleucine synthesis. LEU2-d is a low expression version of LEU2 thatselects for increased copy number (e.g., gene or plasmid copy number,for example) to allow survival on minimal media without leucine. LYS2encodes an activity involved in lysine synthesis, and allows counterselection for recombination out of the LYS2 gene using alpha-aminoadipate (-amino adipate).

Dominant selectable markers are useful because they also allowindustrial and/or prototrophic strains to be used for geneticmanipulations. Additionally, dominant selectable markers provide theadvantage that rich medium can be used for plating and culture growth,and thus growth rates are markedly increased. Non-limiting examples ofdominant selectable markers include; Tn903 kan^(r), Cm^(r), Hyg^(r),CUP1, and DHFR. Tn903 kan^(r) encodes an activity involved in kanamycinantibiotic resistance (e.g., typically neomycin phosphotransferase II orNPTII, for example). Cm^(r) encodes an activity involved inchloramphenicol antibiotic resistance (e.g., typically chloramphenicolacetyl transferase or CAT, for example). Hyg^(r) encodes an activityinvolved in hygromycin resistance by phosphorylation of hygromycin B(e.g., hygromycin phosphotransferase, or HPT). CUP1 encodes an activityinvolved in resistance to heavy metal (e.g., copper, for example)toxicity. DHFR encodes a dihydrofolate reductase activity which confersresistance to methotrexate and sulfanilamde compounds.

In contrast to site-directed or specific mutagenesis, random mutagenesisdoes not require any sequence information and can be accomplished by anumber of widely different methods. Random mutagenesis often is used tocreate mutant libraries that can be used to screen for the desiredgenotype or phenotype. Non-limiting examples of random mutagenesisinclude; chemical mutagenesis, UV-induced mutagenesis, insertion elementor transposon-mediated mutagenesis, DNA shuffling, error-prone PCRmutagenesis, and the like.

Chemical mutagenesis often involves chemicals like ethylmethanesulfonate (EMS), nitrous acid, mitomycin C,N-methyl-N-nitrosourea (MNU), diepoxybutane (DEB), 1, 2, 7,8-diepoxyoctane (DEO), methyl methane sulfonate (MMS),N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide(4-NQO),2-methyloxy-6-chloro-9(3-[ethyl-2-chloroethyl]-aminopropylamino)-acridinedihydrochloride(ICR-170), 2-amino purine (2AP), and hydroxylamine (HA), provided hereinas non-limiting examples. These chemicals can cause base-pairsubstitutions, frameshift mutations, deletions, transversion mutations,transition mutations, incorrect replication, and the like. In someembodiments, the mutagenesis can be carried out in vivo. Sometimes themutagenic process involves the use of the host organisms DNA replicationand repair mechanisms to incorporate and replicate the mutagenized baseor bases.

Another type of chemical mutagenesis involves the use of base-analogs.The use of base-analogs cause incorrect base pairing which in thefollowing round of replication is corrected to a mismatched nucleotidewhen compared to the starting sequence. Base analog mutagenesisintroduces a small amount of non-randomness to random mutagenesis,because specific base analogs can be chose which can be incorporated atcertain nucleotides in the starting sequence. Correction of themispairing typically yields a known substitution. For example,Bromo-deoxyuridine (BrdU) can be incorporated into DNA and replaces T inthe sequence. The host DNA repair and replication machinery can sometimecorrect the defect, but sometimes will mispair the BrdU with a G. Thenext round of replication then causes a G-C transversion from theoriginal A-T in the native sequence.

Ultra violet (UV) induced mutagenesis is caused by the formation ofthymidine dimers when UV light irradiates chemical bonds between twoadjacent thymine residues. Excision repair mechanism of the hostorganism correct the lesion in the DNA, but occasionally the lesion isincorrectly repaired typically resulting in a C to T transition.

Insertion element or transposon-mediated mutagenesis makes use ofnaturally occurring or modified naturally occurring mobile geneticelements. Transposons often encode accessory activities in addition tothe activities necessary for transposition (e.g., movement using atransposase activity, for example). In many examples, transposonaccessory activities are antibiotic resistance markers (e.g., see Tn903kan^(r) described above, for example). Insertion elements typically onlyencode the activities necessary for movement of the nucleic acidsequence. Insertion element and transposon mediated mutagenesis oftencan occur randomly, however specific target sequences are known for sometransposons. Mobile genetic elements like IS elements or Transposons(Tn) often have inverted repeats, direct repeats or both inverted anddirect repeats flanking the region coding for the transposition genes.Recombination events catalyzed by the transposase cause the element toremove itself from the genome and move to a new location, leaving behinda portion of an inverted or direct repeat. Classic examples oftransposons are the “mobile genetic elements” discovered in maize.Transposon mutagenesis kits are commercially available which aredesigned to leave behind a 5 codon insert (e.g., Mutation GenerationSystem kit, Finnzymes, World Wide Web URL finnzymes.us, for example).This allows the artisan to identify the insertion site, without fullydisrupting the function of most genes.

DNA shuffling is a method which uses DNA fragments from members of amutant library and reshuffles the fragments randomly to generate newmutant sequence combinations. The fragments are typically generatedusing DNaseI, followed by random annealing and re-joining using selfpriming PCR. The DNA overhanging ends, from annealing of randomfragments, provide “primer” sequences for the PCR process. Shuffling canbe applied to libraries generated by any of the above mutagenesismethods.

Error prone PCR and its derivative rolling circle error prone PCR usesincreased magnesium and manganese concentrations in conjunction withlimiting amounts of one or two nucleotides to reduce the fidelity of theTaq polymerase. The error rate can be as high as 2% under appropriateconditions, when the resultant mutant sequence is compared to the wildtype starting sequence. After amplification, the library of mutantcoding sequences must be cloned into a suitable plasmid. Although pointmutations are the most common types of mutation in error prone PCR,deletions and frameshift mutations are also possible. There are a numberof commercial error-prone PCR kits available, including those fromStratagene and Clontech (e.g., World Wide Web URL strategene.com andWorld Wide Web URL clontech.com, respectively, for example). Rollingcircle error-prone PCR is a variant of error-prone PCR in whichwild-type sequence is first cloned into a plasmid, then the wholeplasmid is amplified under error-prone conditions.

As noted above, organisms with altered activities can also be isolatedusing genetic selection and screening of organisms challenged onselective media or by identifying naturally occurring variants fromunique environments. For example, 2-Deoxy-D-glucose is a toxic glucoseanalog. Growth of yeast on this substance yields mutants that areglucose-deregulated. A number of mutants have been isolated using2-Deoxy-D-glucose including transport mutants, and mutants that fermentglucose and galactose simultaneously instead of glucose first thengalactose when glucose is depleted. Similar techniques have been used toisolate mutant microorganisms that can metabolize plastics (e.g., fromlandfills), petrochemicals (e.g., from oil spills), and the like, eitherin a laboratory setting or from unique environments.

Similar methods can be used to isolate naturally occurring mutations ina desired activity when the activity exists at a relatively low ornearly undetectable level in the organism of choice, in someembodiments. The method generally consists of growing the organism to aspecific density in liquid culture, concentrating the cells, and platingthe cells on various concentrations of the substance to which anincrease in metabolic activity is desired. The cells are incubated at amoderate growth temperature, for 5 to 10 days. To enhance the selectionprocess, the plates can be stored for another 5 to 10 days at a lowtemperature. The low temperature sometimes can allow strains that havegained or increased an activity to continue growing while other strainsare inhibited for growth at the low temperature. Following the initialselection and secondary growth at low temperature, the plates can bereplica plated on higher or lower concentrations of the selectionsubstance to further select for the desired activity.

A native, heterologous or mutagenized polynucleotide can be introducedinto a nucleic acid reagent for introduction into a host organism,thereby generating an engineered microorganism. Standard recombinant DNAtechniques (restriction enzyme digests, ligation, and the like) can beused by the artisan to combine the mutagenized nucleic acid of interestinto a suitable nucleic acid reagent capable of (i) being stablymaintained by selection in the host organism, or (ii) being integratinginto the genome of the host organism. As noted above, sometimes nucleicacid reagents comprise two replication origins to allow the same nucleicacid reagent to be manipulated in bacterial before final introduction ofthe final product into the host organism (e.g., yeast or fungus forexample). Standard molecular biology and recombinant DNA methods areknown (e.g., described in Maniatis, T., E. F. Fritsch and J. Sambrook(1982) Molecular Cloning: a Laboratory Manual; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

Nucleic acid reagents can be introduced into microorganisms usingvarious techniques. Non-limiting examples of methods used to introduceheterologous nucleic acids into various organisms include;transformation, transfection, transduction, electroporation,ultrasound-mediated transformation, particle bombardment and the like.In some instances the addition of carrier molecules (e.g.,bis-benzimdazolyl compounds, for example, see U.S. Pat. No. 5,595,899)can increase the uptake of DNA in cells typically thought to bedifficult to transform by conventional methods. Conventional methods oftransformation are known (e.g., described in Maniatis, T., E. F. Fritschand J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Feedstocks, Media, Supplements & Additives

Engineered microorganisms often are cultured under conditions thatoptimize yield of a fatty dicarboxylic acid (e.g., an eight toeighteen-carbon fatty dicarboxylic acid). Non-limiting examples of fattydicarboxylic acids include suberic acid (i.e., octanedioic acid,1,8-octanedioic acid, octanedioic acid, octane-1,8-dioic acid,1,6-hexanedicarboxylic acid, capryllic diacids), sebacic acid (i.e.,1,10-decanedioic acid, decanedioic acid, decane-1,10-dioic acid,1,8-octanedicarboxylic acid, capric diacid), dodecanedioic acid (i.e.,DDDA, 1,12-dodecanedioic acid, dodecanedioic acid, dodecane-1,12-dioicacid, 1,10-decanedicarboxylic acid, decamethylenedicaboxylic acid,1,10-dicarboxydecane, lauric diacid), tetradecanedioic acid (i.e., TDDA,1,14-tetradecanedioic acid, tetradecanedioic acid,tetradecane-1,14-dioic acid, 1,12-dodecanedicarboxylic acid, myristicdiacid), thapsic acid (i.e., hexadecanedioic acid, 1,16-hexadecanedioicacid, hexadecanedioic acid, hexadecane-1,16-dioic acid,1,14-tetradecanedicarboxylic acid, palmitic diacid),cis-9-hexadecenedioic acid (i.e., palmitoleic diacids), octanedioic acid(i.e., 1,18-octadecanedioic acid, octadecanedioic acid,octadecane-1,18-dioic acid, 1,16-hexadecanedicarboxylic acid, stearicdiacid), cis-9-octadecenedioic acid (i.e., oleic diacids),cis-9,12-octadecenedioic acid (i.e., linoleic diacids),cis-9,12,15-octadecenedioic acid (i.e., linolenic diacids), arachidicdiacid (i.e., eicosanoic diacid, icosanoic diacid), 11-eicosenoic diacid(i.e., cis-11-eicosenedioic acid), 13-eicosenoic diacids (i.e.,cis-13-eicosenedioic acid), arachidonic diacid (i.e.,cis-5,8,11,14-eicosatetraenedioic acid). Culture conditions oftenoptimize activity of one or more of the following activities: omega oxofatty acid dehydrogenase activity, omega hydroxyl fatty aciddehydrogenase activity, acetyl CoA carboxylase activity, monooxygenaseactivity, monooxygenase reductase activity, fatty alcohol oxidase,acyl-CoA ligase, acyl-CoA oxidase, enoyl-CoA hydratase,3-hydroxyacyl-CoA dehydrogenase, and/or acyltransferase (e.g.,acetyl-CoA C-acyltransferase) activities. In general, non-limitingexamples of conditions that may be optimized include the type and amountof carbon source, the type and amount of nitrogen source, thecarbon-to-nitrogen ratio, the oxygen level, growth temperature, pH,length of the biomass production phase, length of target productaccumulation phase, and time of cell harvest.

Culture media generally contain a suitable carbon source. Carbon sourcesuseful for culturing microorganisms and/or fermentation processessometimes are referred to as feedstocks. The term “feedstock” as usedherein refers to a composition containing a carbon source that isprovided to an organism, which is used by the organism to produce energyand metabolic products useful for growth. A feedstock may be a naturalsubstance, a “man-made substance,” a purified or isolated substance, amixture of purified substances, a mixture of unpurified substances orcombinations thereof. A feedstock often is prepared by and/or providedto an organism by a person, and a feedstock often is formulated prior toadministration to the organism. A carbon source may comprise, but is notlimited to including, one or more of the following substances: alkanes,alkenes, mono-carboxylic acids, di-carboxylic acids, monosaccharides(e.g., also referred to as “saccharides,” which include 6-carbon sugars(e.g., glucose, fructose), 5-carbon sugars (e.g., xylose and otherpentoses) and the like), disaccharides (e.g., lactose, sucrose),oligosaccharides (e.g., glycans, homopolymers of a monosaccharide),polysaccharides (e.g., starch, cellulose, heteropolymers ofmonosaccharides or mixtures thereof), sugar alcohols (e.g., glycerol),and renewable feedstocks (e.g., cheese whey permeate, cornsteep liquor,sugar beet molasses, barley malt).

Carbon sources also can be selected from one or more of the followingnon-limiting examples: paraffin (e.g., saturated paraffin, unsaturatedparaffin, substituted paraffin, linear paraffin, branched paraffin, orcombinations thereof); alkanes (e.g., dodecane), alkenes or alkynes,each of which may be linear, branched, saturated, unsaturated,substituted or combinations thereof (described in greater detail below);linear or branched alcohols (e.g., dodecanol); fatty acids (e.g., about1 carbon to about 60 carbons, including free fatty acids, soap stock,for example); esters of fatty acids; monoglycerides; diglycerides;triglycerides, phospholipids. Non-limiting commercial sources ofproducts for preparing feedstocks include plants, plant oils or plantproducts (e.g., vegetable oils (e.g., almond oil, canola oil, cocoabutter, coconut oil, corn oil, cottonseed oil, flaxseed oil, grape seedoil, illipe, olive oil, palm oil, palm olein, palm kernel oil, saffloweroil, peanut oil, soybean oil, sesame oil, shea nut oil, sunflower oilwalnut oil, the like and combinations thereof) and animal fats (e.g.,beef tallow, butterfat, lard, cod liver oil). A carbon source mayinclude a petroleum product and/or a petroleum distillate (e.g., diesel,fuel oils, gasoline, kerosene, paraffin wax, paraffin oil,petrochemicals). In some embodiments, a feedstock comprises petroleumdistillate. A carbon source can be a fatty acid distillate (e.g., a palmoil distillate or corn oil distillate). Fatty acid distillates can beby-products from the refining of crude plant oils. In some embodiments,a feedstock comprises a fatty acid distillate.

In some embodiments, a feedstock comprises a soapstock (i.e. soapstock). A widely practiced method for purifying crude vegetable oils foredible use is the alkali or caustic refining method. This processemploys a dilute aqueous solution of caustic soda to react with the freefatty acids present which results in the formation of soaps. The soapstogether with hydrated phosphatides, gums and prooxidant metals aretypically separated from the refined oil as the heavy phase dischargefrom the refining centrifuge and are typically known as soapstock.

A carbon source also may include a metabolic product that can be useddirectly as a metabolic substrate in an engineered pathway describedherein, or indirectly via conversion to a different molecule usingengineered or native biosynthetic pathways in an engineeredmicroorganism. In certain embodiments, metabolic pathways can bepreferentially biased towards production of a desired product byincreasing the levels of one or more activities in one or more metabolicpathways having and/or generating at least one common metabolic and/orsynthetic substrate. In some embodiments, a metabolic byproduct (e.g.,fatty acid) of an engineered activity (e.g., omega oxidation activity)can be used in one or more metabolic pathways selected fromgluconeogenesis, pentose phosphate pathway, glycolysis, fatty acidsynthesis, beta oxidation, and omega oxidation, to generate a carbonsource that can be converted to a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid).

The term “paraffin” as used herein refers to the common name for alkanehydrocarbons, independent of the source (e.g., plant derived, petroleumderived, chemically synthesized, fermented by a microorganism), orcarbon chain length. A carbon source sometimes comprises a paraffin, andin some embodiments, a paraffin is predominant in a carbon source (e.g.,about 75%, 80%, 85%, 90% or 95% paraffin). A paraffin sometimes issaturated (e.g., fully saturated), sometimes includes one or moreunsaturations (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 unsaturations)and sometimes is substituted with one or more non-hydrogen substituents.Non-limiting examples of non-hydrogen substituents include halo, acetyl,═O, ═N—CN, ═N—OR, ═NR, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂,NRCOOR, NRCOR, CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R isindependently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R isoptionally substituted with halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C8alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl orC5-C10 heteroaryl.

In some embodiments a feedstock is selected according to the genotypeand/or phenotype of the engineered microorganism to be cultured. Forexample, a feedstock rich in 12-carbon fatty acids, 12-carbondicarboxylic acids or 12-carbon paraffins, or a mixture of 10, 12 and14-carbon compounds can be useful for culturing yeast strains harboringan alteration that partially blocks beta oxidation by disrupting POX4activity, as described herein. Non-limiting examples of carbon sourceshaving 10 to 14 carbons include fats (e.g., coconut oil, palm kerneloil), paraffins (e.g., alkanes, alkenes, or alkynes) having 10 to 14carbons, (e.g., dodecane (also referred to as adakane12, bihexyl,dihexyl and duodecane); tetradecane), alkene and alkyne derivatives),fatty acids (dodecanoic acid, tetradecanoic acid), fatty alcohols(dodecanol, tetradecanol), the like, non-toxic substituted derivativesor combinations thereof.

A carbon source sometimes comprises an alkyl, alkenyl or alkynylcompound or molecule (e.g., a compound that includes an alkyl, alkenylor alkynyl moiety (e.g., alkane, alkene, alkyne)). In certainembodiments, an alkyl, alkenyl or alkynyl molecule, or combinationthereof, is predominant in a carbon source (e.g., about 75%, 80%, 85%,90% or 95% of such molecules). As used herein, the terms “alkyl,”“alkenyl” and “alkynyl” include straight-chain (referred to herein as“linear”), branched-chain (referred to herein as “non-linear”), cyclicmonovalent hydrocarbyl radicals, and combinations of these, whichcontain only C and H atoms when they are unsubstituted. Non-limitingexamples of alkyl moieties include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. An alkyl thatcontains only C and H atoms and is unsubstituted sometimes is referredto as “saturated.” An alkenyl or alkynyl generally is “unsaturated” asit contains one or more double bonds or triple bonds, respectively. Analkenyl can include any number of double bonds, such as 1, 2, 3, 4 or 5double bonds, for example. An alkynyl can include any number of triplebonds, such as 1, 2, 3, 4 or 5 triple bonds, for example. Alkyl, alkenyland alkynyl molecules sometimes contain between about 2 to about 60carbon atoms (C). For example, an alkyl, alkenyl and alkynyl moleculecan include about 1 carbon atom, about 2 carbon atoms, about 3 carbonatoms, about 4 carbon atoms, about 5 carbon atoms, about 6 carbon atoms,about 7 carbon atoms, about 8 carbon atoms, about 9 carbon atoms, about10 carbon atoms, about 12 carbon atoms, about 14 carbon atoms, about 16carbon atoms, about 18 carbon atoms, about 20 carbon atoms, about 22carbon atoms, about 24 carbon atoms, about 26 carbon atoms, about 28carbon atoms, about 30 carbon atoms, about 32 carbon atoms, about 34carbon atoms, about 36 carbon atoms, about 38 carbon atoms, about 40carbon atoms, about 42 carbon atoms, about 44 carbon atoms, about 46carbon atoms, about 48 carbon atoms, about 50 carbon atoms, about 52carbon atoms, about 54 carbon atoms, about 56 carbon atoms, about 58carbon atoms or about 60 carbon atoms. In some embodiments, paraffinscan have a mean number of carbon atoms of between about 8 to about 18carbon atoms (e.g., about 8 carbon atoms, about 9 carbon atoms, about 10carbon atoms, about 11 carbon atoms, about 12 carbon atoms, about 13carbon atoms, about 14 carbon atoms, about 15 carbon atoms, about 16carbon atoms, about 17 carbon atoms and about 18 carbon atoms). A singlegroup can include more than one type of multiple bond, or more than onemultiple bond. Such groups are included within the definition of theterm “alkenyl” when they contain at least one carbon-carbon double bond,and are included within the term “alkynyl” when they contain at leastone carbon-carbon triple bond. Alkyl, alkenyl and alkynyl moleculesinclude molecules that comprise an alkyl, alkenyl and/or alkynyl moiety,and include molecules that consist of an alkyl, alkenyl or alkynylmoiety (i.e., alkane, alkene and alkyne molecules).

Alkyl, alkenyl and alkynyl substituents sometimes contain 1-20C (alkyl)or 2-20C (alkenyl or alkynyl). They can contain about 8-20C or about10-20C in some embodiments. A single group can include more than onetype of multiple bond, or more than one multiple bond. Such groups areincluded within the definition of the term “alkenyl” when they containat least one carbon-carbon double bond, and are included within the term“alkynyl” when they contain at least one carbon-carbon triple bond.

Alkyl, alkenyl and alkynyl groups or compounds sometimes are substitutedto the extent that such substitution can be synthesized and can exist.Typical substituents include, but are not limited to, halo, acetyl, ═O,═N—CN, ═N—OR, ═NR, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR,NRCOR, CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R isindependently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8heteroalkynyl, C6-C11 aryl, or 05-C11 heteroaryl, and each R isoptionally substituted with halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′₂,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C8alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl orC5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also besubstituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10heteroaryl, each of which can be substituted by the substituents thatare appropriate for the particular group.

“Acetylene” or “acetyl” substituents are 2-10C alkynyl groups that areoptionally substituted, and are of the formula —C≡C—Ri, where Ri is H orC1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl,C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl,and each Ri group is optionally substituted with one or moresubstituents selected from halo, ═O, ═N—CN, ═N—OR′, ═NR′, OR′, NR′2,SR′, SO₂R′, SO₂NR′₂, NR′SO₂R′, NR′CONR′₂, NR′COOR′, NR′COR′, CN, COOR′,CONR′₂, OOCR′, COR′, and NO₂, where each R′ is independently H, C1-C6alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl,C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each ofwhich is optionally substituted with one or more groups selected fromhalo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl,hydroxy, amino, and ═O; and where two R′ can be linked to form a 3-7membered ring optionally containing up to three heteroatoms selectedfrom N, O and S. In some embodiments, Ri of —C≡C—Ri is H or Me.

A carbon source sometimes comprises a heteroalkyl, heteroalkenyl and/orheteroalkynyl molecule or compound (e.g., comprises heteroalkyl,heteroalkenyl and/or heteroalkynyl moiety (e.g., heteroalkane,heteroalkene or heteroalkyne)). “Heteroalkyl”, “heteroalkenyl”, and“heteroalkynyl” and the like are defined similarly to the correspondinghydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the ‘hetero’ termsrefer to groups that contain one to three O, S or N heteroatoms orcombinations thereof within the backbone; thus at least one carbon atomof a corresponding alkyl, alkenyl, or alkynyl group is replaced by oneof the specified heteroatoms to form a heteroalkyl, heteroalkenyl, orheteroalkynyl group. The typical and sizes for heteroforms of alkyl,alkenyl and alkynyl groups are generally the same as for thecorresponding hydrocarbyl groups, and the substituents that may bepresent on the heteroforms are the same as those described above for thehydrocarbyl groups. For reasons of chemical stability, it is alsounderstood that, unless otherwise specified, such groups do not includemore than two contiguous heteroatoms except where an oxo group ispresent on N or S as in a nitro or sulfonyl group.

The term “alkyl” as used herein includes cycloalkyl and cycloalkylalkylgroups and compounds, the term “cycloalkyl” may be used herein todescribe a carbocyclic non-aromatic compound or group that is connectedvia a ring carbon atom, and “cycloalkylalkyl” may be used to describe acarbocyclic non-aromatic compound or group that is connected to amolecule through an alkyl linker. Similarly, “heterocyclyl” may be usedto describe a non-aromatic cyclic group that contains at least oneheteroatom as a ring member and that is connected to the molecule via aring atom, which may be C or N; and “heterocyclylalkyl” may be used todescribe such a group that is connected to another molecule through alinker. The sizes and substituents that are suitable for the cycloalkyl,cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the sameas those described above for alkyl groups. As used herein, these termsalso include rings that contain a double bond or two, as long as thering is not aromatic.

A carbon source sometimes comprises an acyl compound or moiety (e.g.,compound comprising an acyl moiety). As used herein, “acyl” encompassesgroups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyl radicalattached at one of the two available valence positions of a carbonylcarbon atom, and heteroacyl refers to the corresponding groups where atleast one carbon other than the carbonyl carbon has been replaced by aheteroatom chosen from N, O and S. Thus heteroacyl includes, forexample, —C(═O)OR and —C(═O)NR₂ as well as —C(═O)-heteroaryl.

Acyl and heteroacyl groups are bonded to any group or molecule to whichthey are attached through the open valence of the carbonyl carbon atom.Typically, they are C1-C8 acyl groups, which include formyl, acetyl,pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which includemethoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups,aryl groups, and heteroforms of such groups that comprise an acyl orheteroacyl group can be substituted with the substituents describedherein as generally suitable substituents for each of the correspondingcomponent of the acyl or heteroacyl group.

A carbon source sometimes comprises one or more aromatic moieties and/orheteroaromatic moieties. “Aromatic” moiety or “aryl” moiety refers to amonocyclic or fused bicyclic moiety having the well-knowncharacteristics of aromaticity; examples include phenyl and naphthyl.Similarly, “heteroaromatic” and “heteroaryl” refer to such monocyclic orfused bicyclic ring systems which contain as ring members one or moreheteroatoms selected from O, S and N. The inclusion of a heteroatompermits aromaticity in 5 membered rings as well as 6 membered rings.Typical heteroaromatic systems include monocyclic C5-C6 aromatic groupssuch as pyridyl, pyrimidyl, pyrazinyl, thienyl, furanyl, pyrrolyl,pyrazolyl, thiazolyl, oxazolyl, and imidazolyl and the fused bicyclicmoieties formed by fusing one of these monocyclic groups with a phenylring or with any of the heteroaromatic monocyclic groups to form aC8-C10 bicyclic group such as indolyl, benzimidazolyl, indazolyl,benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl,pyrazolopyridyl, quinazolinyl, quinoxalinyl, cinnolinyl, and the like.Any monocyclic or fused ring bicyclic system which has thecharacteristics of aromaticity in terms of electron distributionthroughout the ring system is included in this definition. It alsoincludes bicyclic groups where at least the ring which is directlyattached to the remainder of the molecule has the characteristics ofaromaticity. Typically, the ring systems contain 5-12 ring member atoms.The monocyclic heteroaryls sometimes contain 5-6 ring members, and thebicyclic heteroaryls sometimes contain 8-10 ring members.

Aryl and heteroaryl moieties may be substituted with a variety ofsubstituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12aryl, C1-C8 acyl, and heteroforms of these, each of which can itself befurther substituted; other substituents for aryl and heteroaryl moietiesinclude halo, OR, NR₂, SR, SO₂R, SO₂NR₂, NRSO₂R, NRCONR₂, NRCOOR, NRCOR,CN, COOR, CONR₂, OOCR, COR, and NO₂, where each R is independently H,C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl,C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C5-C10 heteroaryl,C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each R is optionallysubstituted as described above for alkyl groups. The substituent groupson an aryl or heteroaryl group may be further substituted with thegroups described herein as suitable for each type of such substituentsor for each component of the substituent. Thus, for example, anarylalkyl substituent may be substituted on the aryl portion withsubstituents typical for aryl groups, and it may be further substitutedon the alkyl portion with substituents as typical or suitable for alkylgroups.

Similarly, “arylalkyl” and “heteroarylalkyl” refer to aromatic andheteroaromatic ring systems, which are stand-alone molecules (e.g.,benzene or substituted benzene, pyridine or substituted pyridine), orwhich are bonded to an attachment point through a linking group such asan alkylene, including substituted or unsubstituted, saturated orunsaturated, cyclic or acyclic linkers. A linker often is C1-C8 alkyl ora hetero form thereof. These linkers also may include a carbonyl group,thus making them able to provide substituents as an acyl or heteroacylmoiety. An aryl or heteroaryl ring in an arylalkyl or heteroarylalkylgroup may be substituted with the same substituents described above foraryl groups. An arylalkyl group sometimes includes a phenyl ringoptionally substituted with the groups defined above for aryl groups anda C1-C4 alkylene that is unsubstituted or is substituted with one or twoC1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkylgroups can optionally cyclize to form a ring such as cyclopropane,dioxolane, or oxacyclopentane. Similarly, a heteroarylalkyl group oftenincludes a C5-C6 monocyclic heteroaryl group optionally substituted withone or more of the groups described above as substituents typical onaryl groups and a C1-C4 alkylene that is unsubstituted. Aheteroarylalkyl group sometimes is substituted with one or two C1-C4alkyl groups or heteroalkyl groups, or includes an optionallysubstituted phenyl ring or C5-C6 monocyclic heteroaryl and a C1-C4heteroalkylene that is unsubstituted or is substituted with one or twoC1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groupscan optionally cyclize to form a ring such as cyclopropane, dioxolane,or oxacyclopentane.

Where an arylalkyl or heteroarylalkyl group is described as optionallysubstituted, the substituents may be on the alkyl or heteroalkyl portionor on the aryl or heteroaryl portion of the group. The substituentsoptionally present on the alkyl or heteroalkyl portion sometimes are thesame as those described above for alkyl groups, and the substituentsoptionally present on the aryl or heteroaryl portion often are the sameas those described above for aryl groups generally.

“Arylalkyl” groups as used herein are hydrocarbyl groups if they areunsubstituted, and are described by the total number of carbon atoms inthe ring and alkylene or similar linker. Thus a benzyl group is aC7-arylalkyl group, and phenylethyl is a C8-arylalkyl.

“Heteroarylalkyl” as described above refers to a moiety comprising anaryl group that is attached through a linking group, and differs from“arylalkyl” in that at least one ring atom of the aryl moiety or oneatom in the linking group is a heteroatom selected from N, O and S. Theheteroarylalkyl groups are described herein according to the totalnumber of atoms in the ring and linker combined, and they include arylgroups linked through a heteroalkyl linker; heteroaryl groups linkedthrough a hydrocarbyl linker such as an alkylene; and heteroaryl groupslinked through a heteroalkyl linker. Thus, for example,C7-heteroarylalkyl includes pyridylmethyl, phenoxy, andN-pyrrolylmethoxy.

“Alkylene” as used herein refers to a divalent hydrocarbyl group.Because an alkylene is divalent, it can link two other groups together.An alkylene often is referred to as —(CH₂)_(n)— where n can be 1-20,1-10, 1-8, or 1-4, though where specified, an alkylene can also besubstituted by other groups, and can be of other lengths, and the openvalences need not be at opposite ends of a chain. Thus —CH(Me)- and—C(Me)₂—may also be referred to as alkylenes, as can a cyclic group suchas cyclopropan-1,1-diyl. Where an alkylene group is substituted, thesubstituents include those typically present on alkyl groups asdescribed herein.

In some embodiments, a feedstock includes a mixture of carbon sources,where each carbon source in the feedstock is selected based on thegenotype of the engineered microorganism. In certain embodiments, amixed carbon source feedstock includes one or more carbon sourcesselected from sugars, cellulose, alkanes, fatty acids,triacylglycerides, paraffins, the like and combinations thereof.

Nitrogen may be supplied from an inorganic (e.g.,(NH.sub.4).sub.2SO.sub.4) or organic source (e.g., urea or glutamate).In addition to appropriate carbon and nitrogen sources, culture mediaalso can contain suitable minerals, salts, cofactors, buffers, vitamins,metal ions (e.g., Mn.sup.+2, Co.sup.+2, Zn.sup.+2, Mg.sup.+2) and othercomponents suitable for culture of microorganisms.

Engineered microorganisms sometimes are cultured in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)). In some embodiments,engineered microorganisms are cultured in a defined minimal media thatlacks a component necessary for growth and thereby forces selection of adesired expression cassette (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Culture media in some embodiments are common commercially preparedmedia, such as Yeast Nitrogen Base (DIFCO Laboratories, Detroit, Mich.).Other defined or synthetic growth media may also be used and theappropriate medium for growth of the particular microorganism are known.A variety of host organisms can be selected for the production ofengineered microorganisms. Non-limiting examples include yeast (e.g.,Candida tropicalis (e.g., ATCC20336, ATCC20913, ATCC20962), Yarrowialipolytica (e.g., ATCC20228)) and filamentous fungi (e.g., Aspergillusnidulans (e.g., ATCC38164) and Aspergillus parasiticus (e.g., ATCC24690)). In specific embodiments, yeast are cultured in YPD media (10g/L Bacto Yeast Extract, 20 g/L Bacto Peptone, and 20 g/L Dextrose).Filamentous fungi, in particular embodiments, are grown in CM (CompleteMedium) containing 10 g/L Dextrose, 2 g/L Bacto Peptone, 1 g/L BactoYeast Extract, 1 g/L Casamino acids, 50 mL/L 20× Nitrate Salts (120 g/LNaNO₃, 10.4 g/L KCl, 10.4 g/L MgSO₄.7 H₂O), 1 mL/L 1000× Trace Elements(22 g/L ZnSO₄.7 H₂O, 11 g/L H₃BO₃, 5 g/L MnCl₂.7 H₂O, 5 g/L FeSO₄.7 H₂O,1.7 g/L CoC1₂.6 H₂O, 1.6 g/L CuSO₄.5 H₂O, 1.5 g/L Na₂MoO₄.2 H₂O, and 50g/L Na₄EDTA), and 1 mL/L Vitamin Solution (100 mg each of Biotin,pyridoxine, thiamine, riboflavin, p-aminobenzoic acid, and nicotinicacid in 100 mL water).

Growth Conditions & Fermentation

A suitable pH range for the fermentation often is between about pH 4.0to about pH 8.0, where a pH in the range of about pH 5.5 to about pH 7.0sometimes is utilized for initial culture conditions. Depending on thehost organism, culturing may be conducted under aerobic or anaerobicconditions, where microaerobic conditions sometimes are maintained. Atwo-stage process may be utilized, where one stage promotesmicroorganism proliferation and another state promotes production oftarget molecule. In a two-stage process, the first stage may beconducted under aerobic conditions (e.g., introduction of air and/oroxygen) and the second stage may be conducted under anaerobic conditions(e.g., air or oxygen are not introduced to the culture conditions). Insome embodiments, the first stage may be conducted under anaerobicconditions and the second stage may be conducted under aerobicconditions. In certain embodiments, a two-stage process may include twomore organisms, where one organism generates an intermediate product inone stage and another organism processes the intermediate product into atarget fatty dicarboxylic acid product (e.g., sebacic or dodecanedioicacid) in another stage, for example.

A variety of fermentation processes may be applied for commercialbiological production of a target fatty dicarboxylic acid product. Insome embodiments, commercial production of a target fatty dicarboxylicacid product from a recombinant microbial host is conducted using abatch, fed-batch or continuous fermentation process, for example.

A batch fermentation process often is a closed system where the mediacomposition is fixed at the beginning of the process and not subject tofurther additions beyond those required for maintenance of pH and oxygenlevel during the process. At the beginning of the culturing process themedia is inoculated with the desired organism and growth or metabolicactivity is permitted to occur without adding additional sources (i.e.,carbon and nitrogen sources) to the medium. In batch processes themetabolite and biomass compositions of the system change constantly upto the time the culture is terminated. In a typical batch process, cellsproceed through a static lag phase to a high-growth log phase andfinally to a stationary phase, wherein the growth rate is diminished orhalted. Left untreated, cells in the stationary phase will eventuallydie.

A variation of the standard batch process is the fed-batch process,where the carbon source is continually added to the fermentor over thecourse of the fermentation process. Fed-batch processes are useful whencatabolite repression is apt to inhibit the metabolism of the cells orwhere it is desirable to have limited amounts of carbon source in themedia at any one time. Measurement of the carbon source concentration infed-batch systems may be estimated on the basis of the changes ofmeasurable factors such as pH, dissolved oxygen and the partial pressureof waste gases (e.g., CO.sub.2).

Batch and fed-batch culturing methods are known in the art. Examples ofsuch methods may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, 2.sup.nd ed., (1989) SinauerAssociates Sunderland, Mass. and Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992).

In continuous fermentation process a defined media often is continuouslyadded to a bioreactor while an equal amount of culture volume is removedsimultaneously for product recovery. Continuous cultures generallymaintain cells in the log phase of growth at a constant cell density.Continuous or semi-continuous culture methods permit the modulation ofone factor or any number of factors that affect cell growth or endproduct concentration. For example, an approach may limit the carbonsource and allow all other parameters to moderate metabolism. In somesystems, a number of factors affecting growth may be alteredcontinuously while the cell concentration, measured by media turbidity,is kept constant. Continuous systems often maintain steady state growthand thus the cell growth rate often is balanced against cell loss due tomedia being drawn off the culture. Methods of modulating nutrients andgrowth factors for continuous culture processes, as well as techniquesfor maximizing the rate of product formation, are known and a variety ofmethods are detailed by Brock, supra.

In some embodiments involving fermentation, the fermentation can becarried out using two or more microorganisms (e.g., host microorganism,engineered microorganism, isolated naturally occurring microorganism,the like and combinations thereof), where a feedstock is partially orcompletely utilized by one or more organisms in the fermentation (e.g.,mixed fermentation), and the products of cellular respiration ormetabolism of one or more organisms can be further metabolized by one ormore other organisms to produce a desired target product (e.g., sebacicacid, dodecanedioic acid, hexanoic acid). In certain embodiments, eachorganism can be fermented independently and the products of cellularrespiration or metabolism purified and contacted with another organismto produce a desired target product. In some embodiments, one or moreorganisms are partially or completely blocked in a metabolic pathway(e.g., beta oxidation, omega oxidation, the like or combinationsthereof), thereby producing a desired product that can be used as afeedstock for one or more other organisms. Any suitable combination ofmicroorganisms can be utilized to carry out mixed fermentation orsequential fermentation.

Enhanced Fermentation Processes

It has been determined that certain feedstock components are toxic to,or produce a by-product (e.g., metabolite) that is toxic to, yeastutilized in a fermentation process for the purpose of producing a targetproduct (e.g., a C4 to C24 diacid). A toxic component or metabolite froma feedstock sometimes is utilized by the yeast to produce a targetproduct (e.g., target molecule).

In some instances, a fatty acid component having 12 carbons (i.e., C12)or fewer carbons can be toxic to yeast. Components that are not freefatty acids, but are processed by yeast to a fatty acid having twelve orfewer carbons, also can have a toxic effect. Non-limiting examples ofsuch components are esters of fatty acids (e.g., methyl esters) that areprocessed by yeast into a fatty acid having twelve or fewer carbons.Feedstocks containing molecules that are directly toxic, or indirectlytoxic by conversion of a non-toxic component to a toxic metabolite, arecollectively referred to as “toxic feedstocks” and “toxic components.”Providing yeast with a feedstock that comprises or delivers one or moretoxic components can reduce the viability of the yeast and/or reduce theamount of target product produced by the yeast.

In some embodiments, a process for overcoming the toxic effect ofcertain components in a feedstock includes first inducing yeast with afeedstock not containing a substantially toxic component and thenproviding the yeast with a feedstock that comprises a toxic component.Thus, in some embodiments, provided is a method for producing a diacidby a yeast from a feedstock toxic to the yeast, comprising: (a)contacting a genetically modified yeast in culture with a firstfeedstock comprising a component not substantially toxic to the yeast,thereby performing an induction; and (b) contacting the yeast after theinduction in (a) with a second feedstock that comprises or delivers acomponent toxic to the yeast (“toxic component”), whereby a diacid isproduced by the yeast in an amount greater than the amount of the diacidproduced when the induction is not performed.

A toxic component provided by the second feedstock sometimes isprocessed by the yeast into a target product (e.g., diacid). Sometimes acomponent not substantially toxic to the yeast in the first feedstock(e.g., an inducer) is processed by the yeast into a target product orbyproduct (e.g., diacid containing a different number of carbons thanthe target product). The first feedstock sometimes comprises a componentnot substantially toxic to the yeast having the same number of carbonsas the component in the second feedstock, or a metabolite processed bythe yeast from a component in the second feedstock, that issubstantially toxic to the yeast. In some embodiments, the firstfeedstock comprises a component not substantially toxic to the yeasthaving a different number of carbons as the component in the secondfeedstock, or a metabolite processed by yeast from a component in thesecond feedstock, that is substantially toxic to the yeast. In certainembodiments, the first feedstock comprises a component that is notsubstantially toxic to the yeast (e.g., an inducer) that has the samenumber of carbons as the target product. Sometimes the first feedstockcomprises a component not substantially toxic to the yeast (e.g., aninducer) that has a different number of carbons as the target product(e.g., diacid).

In some embodiments, the first feedstock comprises an ester of a fattyacid that is not substantially toxic to the yeast (e.g., methyl ester),and sometimes the fatty acid has more than 12 carbons. The firstfeedstock sometimes comprises a fatty acid that is not substantiallytoxic to the yeast, and in some cases the fatty acid has more than 12carbons. The first feedstock sometimes comprises a triglyceride, whichtriglyceride often contains various chain-length fatty acids, that isnot substantially toxic to the yeast. In certain cases the firstfeedstock comprises an aliphatic chain, which aliphatic chain oftencontains more than 6 carbons, that is not substantially toxic to theyeast. In some embodiments, the first feedstock comprises one or morealkanes (e.g., linear alkanes, branched alkanes, substituted alkanes)with chain lengths greater than 6 carbons. In some embodiments a targetproduct is a C12 diacid, the first feedstock comprises an alkane (e.g.,alkane inducer) and the second feedstock comprises a C12 fatty acid oran ester of a C12 fatty acid, where the alkane sometimes is a C12alkane. In some embodiments a target product is a 010 diacid, the firstfeedstock comprises an alkane (e.g., alkane inducer) and the secondfeedstock comprises a C10 fatty acid or an ester of a C10 fatty acid,where the alkane sometimes is a 010 alkane. In some embodiments a targetproduct is a C18 diacid, the first feedstock comprises an alkane (e.g.,alkane inducer) and the second feedstock comprises a C18 fatty acid oran ester of a C18 fatty acid, where the alkane sometimes is a C18alkane. In certain embodiments, one or more of the (i) components in thefirst feedstock and/or the second feedstock and (ii) products (e.g.,target product) are saturated. In some embodiments, one or more of the(i) components in the first feedstock and/or the second feedstock and(ii) products (e.g., target product) include one or more unsaturations(e.g., one or more double bonds).

In some embodiments, the second feedstock is provided to the yeast acertain amount of time after the first feedstock is provided to theyeast. The amount of time sometimes is about 1 hour to about 48 hours,sometimes is about 1 hour to about 12 hours (e.g., about 2 hours, 3,hours, 4, hours, 5, hours, 6 hours, 7 hours, 8, hours, 9 hours, 10 hoursor 11 hours), and sometimes is about 3 hours to about 9 hours. In someembodiments, the yeast is a Candida spp. yeast, or another yeastdescribed herein.

Taget Product Production, Isolation and Yield

In various embodiments a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) isisolated or purified from the culture media or extracted from theengineered microorganisms. In some embodiments, fermentation offeedstocks by methods described herein can produce a target fattydicarboxylic acid product (e.g., sebacic or dodecanedioic acid) at alevel of about 10% to about 100% of theoretical yield (e.g., about 15%,about 20%, about 25% or more of theoretical yield (e.g., 25% or more,26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% ormore, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more,37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% ormore, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more,48% or more, 49% or more, 50% or more, 51% or more, 52% or more, 53% ormore, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more,59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% ormore, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more,70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% ormore, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more,81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more, or 99% or more of theoretical yield). The term“theoretical yield” as used herein refers to the amount of product thatcould be made from a starting material if the reaction is 100% complete.Theoretical yield is based on the stoichiometry of a reaction and idealconditions in which starting material is completely consumed, undesiredside reactions do not occur, the reverse reaction does not occur, andthere are no losses in the work-up procedure. Culture media may betested for target product (e.g., sebacic or dodecanedioic acid)concentration and drawn off when the concentration reaches apredetermined level. Detection methods are known in the art, includingbut not limited to chromatographic methods (e.g., gas chromatography) orcombined chromatographic/mass spectrometry (e.g., GC-MS) methods. Targetproduct (e.g., sebacic or dodecanedioic acid) may be present at a rangeof levels as described herein.

A target fatty dicarboxylic acid product sometimes is retained within anengineered microorganism after a culture process is completed, and incertain embodiments, the target product is secreted out of themicroorganism into the culture medium. For the latter embodiments, (i)culture media may be drawn from the culture system and fresh medium maybe supplemented, and/or (ii) target product may be extracted from theculture media during or after the culture process is completed.Engineered microorganisms may be cultured on or in solid, semi-solid orliquid media. In some embodiments media is drained from cells adheringto a plate. In certain embodiments, a liquid-cell mixture is centrifugedat a speed sufficient to pellet the cells but not disrupt the cells andallow extraction of the media, as known in the art. The cells may thenbe resuspended in fresh media. Target product may be purified fromculture media according to known methods know in the art.

In some embodiments, a target diacid is present in a product containingother diacids and/or byproducts. The target diacid can be purified fromthe other diacids and/or byproducts using a suitable purificationprocedure. A partically purified or substantially purified target diacidmay be produced using a purification process.

Provided herein are non-limiting examples of methods useful forrecovering target product from fermentation broth and/orisolating/partially purifying a target fatty dicarboxylic acid productfrom non-target products when utilizing mixed chain length feedstocks.Recovery of a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) fromfermentation broth can be accomplished using a variety of methods.Optionally, one can first employ a centrifugation step to separate cellmass and a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioicacid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) from the aqueous phase. Afatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) has limited solubility inwater under fermentation conditions, and has a density similar to thatof water. Upon centrifugation, the majority of fatty dicarboxylic acid(e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid) will be pulled away from the water stream, and beconcentrated in the cell mass stream. The concentrated fattydicarboxylic acid stream will then be further concentrated viafiltration steps (e.g., solid dodecanedioic acid will be retained on afilter, allowing water to pass through, concentrating the product). Oncethe fatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) is concentrated to the desiredlevel, the temperature will be increased to above its melting point of130° C. After the fatty dicarboxylic acid is melted, the remainingimpurities are removed via filtration; the final product is recovered bydecreasing the temperature, allowing the fatty dicarboxylic acid tosolidify, and collecting the solid product.

Alternatively, a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) can berecovered from fermentation broth by first extracting the broth with anorganic solvent in which a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) issoluble (e.g., ethanol). The organic solvent phase can then be filteredthrough various membranes to further purify the fatty dicarboxylic acid.Subsequent extractions with the same or a different organic solvent canthen be performed and each round of extraction can be followed bymembrane filtration to further concentrate the fatty dicarboxylic acid.The organic solvent can be evaporated, leaving the fatty dicarboxylicacid behind as a residue and the residue can be dried to provide thefatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) in solid form.

In certain embodiments, target product is extracted from the culturedengineered microorganisms. The microorganism cells may be concentratedthrough centrifugation at a speed sufficient to shear the cellmembranes. In some embodiments, the cells may be physically disrupted(e.g., shear force, sonication) or chemically disrupted (e.g., contactedwith detergent or other lysing agent). The phases may be separated bycentrifugation or other method known in the art and target product maybe isolated according to known methods.

Commercial grade target product sometimes is provided in substantiallypure form (e.g., 90% pure or greater, 95% pure or greater, 99% pure orgreater or 99.5% pure or greater). In some embodiments, target productmay be modified into any one of a number of downstream products. Forexample, a fatty dicarboxylic acid (e.g., octanedioic acid, decanedioicacid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) may be polycondensed withhexamethylenediamine to produce nylon. Nylon may be further processedinto fibers for applications in carpeting, automobile tire cord andclothing. A fatty dicarboxylic acid (e.g., octanedioic acid, decanedioicacid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) is also used for manufacturingplasticizers, lubricant components and polyester polyols forpolyurethane systems. Various esters of food grade fatty dicarboxylicacids (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid,tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid,eicosanedioic acid) are used as components in fragrance manufacture,gelling aids, flavorings, acidulant, leavening and buffering agent. Afatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) has two carboxylic acid(—COOH) groups, which can yield two kinds of salts. Its derivatives,acyl halides, anhydrides, esters, amides and nitriles, are used inmaking a variety of downstream products through further reactions ofsubstitution, catalytic reduction, metal hydride reduction, diboranereduction, keto formation with organometallic reagents, electrophilebonding at oxygen, and condensation.

Target product may be provided within cultured microbes containingtarget product, and cultured microbes may be supplied fresh or frozen ina liquid media or dried. Fresh or frozen microbes may be contained inappropriate moisture-proof containers that may also be temperaturecontrolled as necessary. Target product sometimes is provided in culturemedium that is substantially cell-free. In some embodiments targetproduct or modified target product purified from microbes is provided,and target product sometimes is provided in substantially pure form. Incertain embodiments crystallized or powdered target product is provided.Dodecanedioic acid (1,12 dodecanedioic acid; DDDA) is a white powder orcrystal with a melting point of between 260° F. and 266° F. Sebacic acid(1,8 ocatanedicarboxylic acid) is also a white powder or crystal with amelting point of between 268° F. and 274° F. A crystallized or powderedfatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) may be transported in avariety of containers including one ton cartons, drums, 50 pound bagsand the like.

In certain embodiments, a fatty dicarboxylic acid target product (e.g.,dodecanedioic acid or sebacid acid) is produced with a yield of about0.50 grams of target product per gram of feedstock added, or greater;0.51 grams of target product per gram of feedstock added, or greater;0.52 grams of target product per gram of feedstock added, or greater;0.53 grams of target product per gram of feedstock added, or greater;0.54 grams of target product per gram of feedstock added, or greater;0.55 grams of target product per gram of feedstock added, or greater;0.56 grams of target product per gram of feedstock added, or greater;0.57 grams of target product per gram of feedstock added, or greater;0.58 grams of target product per gram of feedstock added, or greater;0.59 grams of target product per gram of feedstock added, or greater;0.60 grams of target product per gram of feedstock added, or greater;0.61 grams of target product per gram of feedstock added, or greater;0.62 grams of target product per gram of feedstock added, or greater;0.63 grams of target product per gram of feedstock added, or greater;0.64 grams of target product per gram of feedstock added, or greater;0.65 grams of target product per gram of feedstock added, or greater;0.66 grams of target product per gram of feedstock added, or greater;0.67 grams of target product per gram of feedstock added, or greater;0.68 grams of target product per gram of feedstock added, or greater;0.69 grams of target product per gram of feedstock added, or greater;0.70 grams of target product per gram of feedstock added or greater;0.71 grams of target product per gram of feedstock added, or greater;0.72 grams of target product per gram of feedstock added, or greater;0.73 grams of target product per gram of feedstock added, or greater;0.74 grams of target product per gram of feedstock added, or greater;0.75 grams of target product per gram of feedstock added, or greater;0.76 grams of target product per gram of feedstock added, or greater;0.77 grams of target product per gram of feedstock added, or greater;0.78 grams of target product per gram of feedstock added, or greater;0.79 grams of target product per gram of feedstock added, or greater;0.80 grams of target product per gram of feedstock added, or greater;0.81 grams of target product per gram of feedstock added, or greater;0.82 grams of target product per gram of feedstock added, or greater;0.83 grams of target product per gram of feedstock added, or greater;0.84 grams of target product per gram of feedstock added, or greater;0.85 grams of target product per gram of feedstock added, or greater;0.86 grams of target product per gram of feedstock added, or greater;0.87 grams of target product per gram of feedstock added, or greater;0.88 grams of target product per gram of feedstock added, or greater;0.89 grams of target product per gram of feedstock added, or greater;0.90 grams of target product per gram of feedstock added, or greater;0.91 grams of target product per gram of feedstock added, or greater;0.92 grams of target product per gram of feedstock added, or greater;0.93 grams of target product per gram of feedstock added, or greater;0.94 grams of target product per gram of feedstock added, or greater;0.95 grams of target product per gram of feedstock added, or greater;0.96 grams of target product per gram of feedstock added, or greater;0.97 grams of target product per gram of feedstock added, or greater;0.98 grams of target product per gram of feedstock added, or greater;0.99 grams of target product per gram of feedstock added, or greater;1.0 grams of target product per gram of feedstock added, or greater; 1.1grams of target product per gram of feedstock added, or greater; 1.2grams of target product per gram of feedstock added, or greater; 1.3grams of target product per gram of feedstock added, or greater; 1.4grams of target product per gram of feedstock added, or greater; orabout 1.5 grams of target product per gram of feedstock added, orgreater.

In certain embodiments, a fatty dicarboxylic acid (e.g., octanedioicacid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) isproduced with a yield of greater than about 0.15 grams per gram of thefeedstock (e.g., dodecane, mixed chain length alkanes, lauric acid,mixed chain length fatty acids, oil, the like or combinations of theforegoing). In some embodiments, a fatty dicarboxylic acid (e.g.,octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioicacid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) isproduced at between about 10% and about 100% of maximum theoreticalyield of any introduced feedstock ((e.g., about 15%, about 20%, about25% or more of theoretical yield (e.g., 25% or more, 26% or more, 27% ormore, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more,33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% ormore, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more,44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% ormore, 50% or more, 51% or more, 52% or more, 53% or more, 54% or more,55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% ormore, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more,66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% ormore, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more,77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% ormore, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more,88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% ormore, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more,or 99% or more of theoretical maximum yield). In certain embodiments, afatty dicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) is produced in a concentrationrange of between about 50 g/L to about 1000 g/L of culture media (e.g.,about 50 g/L, about 55 g/L, about 60 g/L, about 65 g/L, about 70 g/L,about 75 g/L, about 80 g/L, about 85 g/L, about 90 g/L, about 95 g/L,about 100 g/L, about 110 g/L, about 120 g/L, about 130 g/L, about 140g/L, about 150 g/L, about 160 g/L, about 170 g/L, about 180 g/L, about190 g/L, about 200 g/L, about 225 g/L, about 250 g/L, about 275 g/L,about 300 g/L, about 325 g/L, about 350 g/L, about 375 g/L, about 400g/L, about 425 g/L, about 450 g/L, about 475 g/L, about 500 g/L, about550 g/L, about 600 g/L, about 650 g/L, about 700 g/L, about 750 g/L,about 800 g/L, about 850 g/L, about 900 g/L, about 950 g/L, or about1000 g/L).

In some embodiments, a fatty dicarboxylic acid (e.g., octanedioic acid,decanedioic acid, dodecanedioic acid, tetradecanedioic acid,hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid) isproduced at a rate of between about 0.5 g/L/hour to about 5 g/L/hour(e.g., about 0.5 g/L/hour, about 0.6 g/L/hour, about 0.7 g/L/hour, about0.8 g/L/hour, about 0.9 g/L/hour, about 1.0 g/L/hour, about 1.1g/L/hour, about 1.2 g/L/hour, about 1.3 g/L/hour, about 1.4 g/L/hour,about 1.5 g/L/hour, about 1.6 g/L/hour, about 1.7 g/L/hour, about 1.8g/L/hour, about 1.9 g/L/hour, about 2.0 g/L/hour, about 2.25 g/L/hour,about 2.5 g/L/hour, about 2.75 g/L/hour, about 3.0 g/L/hour, about 3.25g/L/hour, about 3.5 g/L/hour, about 3.75 g/L/hour, about 4.0 g/L/hour,about 4.25 g/L/hour, about 4.5 g/L/hour, about 4.75 g/L/hour, or about5.0 g/L/hour.) In certain, embodiments, the engineered organismcomprises between about a 5-fold to about a 500-fold increase in a fattydicarboxylic acid (e.g., octanedioic acid, decanedioic acid,dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid,octadecanedioic acid, eicosanedioic acid) production when compared towild-type or partially engineered organisms of the same strain, underidentical fermentation conditions (e.g., about a 5-fold increase, abouta 10-fold increase, about a 15-fold increase, about a 20-fold increase,about a 25-fold increase, about a 30-fold increase, about a 35-foldincrease, about a 40-fold increase, about a 45-fold increase, about a50-fold increase, about a 55-fold increase, about a 60-fold increase,about a 65-fold increase, about a 70-fold increase, about a 75-foldincrease, about a 80-fold increase, about a 85-fold increase, about a90-fold increase, about a 95-fold increase, about a 100-fold increase,about a 125-fold increase, about a 150-fold increase, about a 175-foldincrease, about a 200-fold increase, about a 250-fold increase, about a300-fold increase, about a 350-fold increase, about a 400-fold increase,about a 450-fold increase, or about a 500-fold increase).

In certain embodiments, the maximum theoretical yield (Y_(max)) ofdodecanedioic acid in a fully beta-oxidation blocked engineeredmicroorganism is about 1.15 grams of dodecanedioic acid produced pergram of lauric acid added. In some embodiments, the maximum theoreticalyield (Y_(max)) of dodecanedioic acid in a fully beta-oxidation blockedengineered microorganism is about 1.07 grams of dodecanedioic acidproduced per gram of methyl laurate added. In certain embodiments, themaximum theoretical yield (Y_(max)) of dodecanedioic acid in a partiallybeta-oxidation blocked engineered microorganism is about 0.82 grams ofdodecanedioic acid produced per gram of oleic acid added. In someembodiments, the maximum theoretical yield (Y_(max)) of dodecanedioicacid in a partially beta-oxidation blocked engineered microorganism isabout 0.95 grams of dodecanedioic acid produced per gram of coconut oiladded. The percentage of Y_(max) for the engineered microorganism underconditions in which dodecanedioic acid is produced is calculated as (%Y_(max))=Y_(p/s)/Y_(max)*100, where (Y_(p/s))=[dodecanedioic acid(g/L)]*final volume of culture in flask (L)]/[feedstock added to flask(g)]. In some embodiments, the engineered microorganism producesdodecanedioic acid at about 10% to about 100% of maximum theoreticalyield.

In certain embodiments, the maximum theoretical yield (Y_(max)) ofsebacic acid in a fully beta-oxidation blocked engineered microorganismis about 1.42 grams of sebacic acid produced per gram of decane added.In some embodiments, the maximum theoretical yield (Y_(max)) of sebacicacid in a fully beta-oxidation blocked engineered microorganism is about1.17 grams of sebacic acid produced per gram of capric acid added. Incertain embodiments, the maximum theoretical yield (Y_(max)) of sebacicacid in a partially beta-oxidation blocked engineered microorganism isabout 0.83 grams of sebacic acid produced per gram of coconut oil added.In some embodiments, the maximum theoretical yield (Y_(max)) of sebacicacid in a partially beta-oxidation blocked engineered microorganism isabout 0.72 grams of sebacic acid produced per gram of oleic acid added.The percentage of Y_(max) for the engineered microorganism underconditions in which sebacic acid is produced is calculated as (%Y_(max))=Y_(p/s)/Y_(max)*100, where (Y_(p/s))=[sebacic acid (g/L)]*finalvolume of culture in flask (L)]/[feedstock added to flask (g)]. In someembodiments, the engineered microorganism produces sebacic acid at about10% to about 100% of maximum theoretical yield.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology. Certain examples set forth below utilize standardrecombinant DNA and other biotechnology protocols known in the art. Manysuch techniques are described in detail in Maniatis, T., E. F. Fritschand J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. DNA mutagenesis canbe accomplished using the Stratagene (San Diego, Calif.) “QuickChange”kit according to the manufacturer's instructions.

Non-limiting examples of recombinant DNA techniques and geneticmanipulation of microorganisms are described herein. In someembodiments, strains of engineered organisms described herein are matedto combine genetic backgrounds to further enhance carbon flux managementthrough native and/or engineered pathways described herein, for theproduction of a desired target product (e.g., sebacic or dodecanedioicacid).

Example 1: Conversion of Decane to Sebacic Acid in Shake FlaskFermentation

50 mL of SP92 medium (6.7 g/L yeast nitrogen base, 3.0 g/L yeastextract, 3.0 g/L (NH₄)₂SO₄, 1.0 g/L K₂HPO₄, 1.0 g/L KH₂PO₄, 75 g/Ldextrose) was inoculated with a single colony of a completelybeta-oxidation blocked strain of Candida tropicalis (ATCC20962) and theculture was grown overnight at 30° C., with shaking at about 300 rpm.Cells were pelleted by centrifugation for 10 minutes at 4° C. and1,050×g and the supernatant discarded. Cells were resuspended in 20 mLTB-low nitrogen (low-N) media (1.7 g/L yeast nitrogen base withoutammonium sulfate, 3.0 g/L yeast extract, 1.0 g/L K₂HPO₄, 1.0 g/L KH₂PO₄)and transferred to a new sterile 250 mL glass baffled flask andincubated at 30° C., with shaking at about 250 rpm, utilizing thefollowing feeding schedule: dextrose fed to 0.1% at 0, 1, 2, 3, 4, and 5hours, dextrose fed to 5% at 30 hours, decane fed to 0.7% at 0, 5, 30,and 48 hours. Samples were removed for gas chromatographic (GC) analysisat 0, 4, 30, and 72 hours. The GC profile showed that the cultureaccumulated the C10 dicarboxylic acid (sebacic acid) with very littleaccumulation of the C10 monocarboxylic acid (capric acid), as shown inFIG. 9. After 72 hours of incubation the concentration of sebacic acidwas 0.94 g/L and the capric acid concentration was 0.01 g/L. There wasno significant accumulation of any other monoacid or diacid.

Example 2: Conversion of Capric Acid to Sebacic Acid in Shake FlaskFermentation

5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base, 3.0 g/L yeastextract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 75 g/Lglycerol) was inoculated with a single colony of Candida tropicalis(ATCC20962) and the culture was grown overnight at 30° C., with shakingat about 250 rpm. This starter culture was then used to inoculate 25 mLcultures in the same medium to an initial OD_(600nm) of 0.4 and grownovernight at 30° C., with shaking at about 300 rpm. Cells were pelletedby centrifugation for 10 minutes at 4° C. and 1,050×g and thesupernatant discarded. Cells were resuspended in 12.5 mL TB-IowNmedia+glycerol (1.7 g/L yeast nitrogen base without ammonium sulfate,3.0 g/L yeast extract, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 75 g/L glycerol)and transferred to a new sterile 250 mL glass baffled flask. Cultureswere fed 0.05% or 0.1% capric acid and incubated at 30° C., with shakingat about 300 rpm. After 24 hours incubation cultures were fed glycerolto 75 g/L and incubation continued before sampling for GC at 48 hours.GC analysis showed that nearly all capric acid was converted to sebacicacid under both starting concentrations of capric acid, as shown in FIG.10.

Example 3: Fermentation Procedure for Conversion of Decane to SebacicAcid

Filter sterilized modified SP92-glycerol fermentation medium (6.7 g/Lyeast nitrogen base, 3.0 g/L yeast extract, 3.0 g/L (NH4)2SO4, 1.0 g/LK2HPO4, 1.0 g/L KH2PO4, 20 g/L glycerol) is transferred to a sterilefermentation vessel. Growth of Candida tropicalis (ATCC20962) isinnoculated to an initial OD_(600nm) of about 1.0 with a 5% inoculum andgrowth carried out under the following conditions: 30° C. with shakingat about 1000 rpm, 1 volume per volume per minute aeration (vvm), pH 5.8and initial volume of 0.3 L. Growth proceeds for approximately 8 hoursand the conversion phase is initiated by the addition of decane to 2g/L. Continuous feeds for decane (1 g/L-h) and glucose (1.5 g/L-h) areinitiated at the same time as the addition of the decane bolus.Fermentation conditions are maintained at 30° C., 1000 rpm, 1 vvm, andpH 5.8 for 44 hours.

Samples were collected for GC analysis at 44 hours after initiating theconversion phase. The data, presented in FIG. 16, shows that the decanewas converted exclusively to the 010 dicarboxylic acid, sebacic acid.Significant evaporative losses from the decane feed bottles prevented anaccurate determination of product yield.

Example 4: Conversion of Mixed Fatty Acid Feedstock to Mixed DiacidProducts Containing Sebacic Acid in Shake Flask Fermentation

5 mL of SP92-glycerol medium (6.7 g/L yeast nitrogen base, 3.0 g/L yeastextract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 75 g/Lglycerol) is inoculated with a single colony of Candida tropicalis(ATCC20962) and grown as described in Example 2. 25 mL of the same mediais inoculated using overnight cultures to an initial OD_(600nm) of 0.4and grown overnight at 30° C., with shaking at about 300 rpm. Cells arepelleted by centrifugation for 10 minutes at 4° C. and 1,050×g and thesupernatant discarded. Cells are resuspended in 12.5 mL TB-IowN mediawithout carbon source (1.7 g/L yeast nitrogen base without ammoniumsulfate, 3.0 g/L yeast extract, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4) andtransferred to a new sterile 250 mL glass baffled flask. Cultures arefed 0.05% capric acid, 0.05% methyl laurate, and 30 g/L glycerol andincubated at 30° C., 300 rpm. After 24 hours of incubation cultures aresampled for GC analysis.

The results, presented in FIG. 17, show that the C12 and C10 fatty acidswere converted to dicarboxylic acids of the same chain length (e.g., C12and C10 dicarboxylic acids), with no evidence of chain shortening of thediacids (e.g., no significant levels of monocarboxylic acids weredetected).

Example 5: Conversion of Long Chain Fatty Acids to Mixed Diacids

SP92 fermentation medium was filter sterilized and transferred to asterile fermentation vessel. Growth of a partially beta-oxidationblocked strain of Candida tropicalis (sAA106) was initiated with a 10%inoculum (initial OD_(600nm)=3.0) and grown under the followingconditions: of 30° C. with shaking at about 1200 rpm, 1 vvm, pH 6.1 andinitial volume of 0.3 L. Growth continued until the glucoseconcentration dropped to less than 2 g/L at which time the conversionphase was initiated by increasing the pH to 8.0 by the addition of 6NKOH and by the addition of methyl myristate to 30 g/L. Immediatelyfollowing the methyl myristate bolus a continuous feed of glucose wasinitiated at a rate of 1.5 g/L-h. Fermentation conditions weremaintained at 30° C., 1200 rpm, 1 vvm, and pH 8.0 for 90 hours withboluses of 30 g/L methyl myristate at 24, 48, and 72 hours afterinitiation of conversion. Samples for GC were collected at 24, 48, 72,and 90 hours. The diacid profile graphically illustrated in FIG. 11shows an accumulation of dicarboxylic acids ranging in chain-length from6 to 14 carbons long, including sebacic acid. The methyl myristatesubstrate (methyl ester of myristic acid) is first converted to the C14dicarboxylic acid via the w-oxidation pathway before being shortened bytwo carbon increments via the cyclic β-oxidation pathway. The glucoseco-feed employed during the fermentation represses the β-oxidationpathway such that all chain-lengths of diacid accumulate. Manipulationof diacid chain-length distribution is being investigated by alteringthe glucose co-feed rate in the fermentation medium, thereby allowinggrowth under varying glucose concentrations.

Example 6: Fermentation Procedure for Conversion of Mixed Long-ChainFatty Acids to Mixed Diacids of Shorter Chain Length

SP92 fermentation medium without glycerol was filter sterilized andtransferred to a sterile fermentation vessel. Autoclaved virgin coconutoil was added to the vessel to a final concentration of 80 g/L. Apartially beta-oxidation blocked Candida tropicalis strain (sAA496) wasinoculated to an initial OD_(600nm) of 1.0 with a 5% inoculum and grownunder the following conditions: 30° C. with shaking at about 1200 rpm, 1vvm, initial pH 6.5 and initial volume of 1.0 L. The effect of pH on thedistribution of fatty acid chain lengths was determined by manipulatingthe pH of the fermentation media. The pH of the fermentation waseither 1) increased to pH 7.5 and controlled at that pH for the entirerun, 2) allowed to drop naturally due to the growth of the culturebefore controlling at pH 6.0 for the rest of the run, or 3) allowed todrop naturally due to the growth of the culture before controlling at pH4.5 for the rest of the run. Samples were collected for GC analysisafter 140 hours of fermentation time. The product diacid composition wasshown to shift to longer chain diacids with increasing pH, as shown inthe table below.

Diacid composition (fraction of total diacids) C12 Diacid Sebacic AcidSuberic Acid Adipic Acid pH 4.5 0.00 0.00 0.68 0.32 pH 6.0 0.03 0.100.75 0.12 pH 7.5 0.16 0.17 0.62 0.05

Example 7: Conversion of Capric Acid to Sebacic Acid in Shake FlaskFermentations Using Fully Beta-Oxidation Blocked Strains HavingAdditional Genetic Modifications in the Omega Oxidation Pathway

Various genetically modified strains of Candida tropicalis wereinoculated into 5 mL of SP92 medium (6.7 g/L yeast nitrogen base, 3.0g/L yeast extract, 3.0 g/L (NH₄)₂SO₄, 1.0 g/L K₂HPO₄, 1.0 g/L KH₂PO₄, 75g/L glycerol). The strains included a completely beta-oxidation blockedstrain of Candida tropicalis (sAA003), as well as derivatives of sAA003with amplified components of the omega-oxidation pathway (e.g., variouscytochrome P450s, cytochrome P450 reductase or combinations thereof) andthe cultures grown overnight at 30° C., with shaking at about 250 rpm.These starter cultures were then used to inoculate 25 mL cultures in thesame medium and grown overnight at 30° C., with shaking at about 250rpm. Cells were pelleted by centrifugation for 10 minutes at 4° C. and1,050×g and the supernatant discarded. Cells were resuspended in 12.5 mLTB-IowN media+glycerol (1.7 g/L yeast nitrogen base without ammoniumsulfate, 3.0 g/L yeast extract, 1.0 g/L K₂HPO₄, 1.0 g/L KH₂PO₄, 75 g/Lglycerol) and transferred to a new sterile 250 mL glass baffled flask.Cultures were fed 0.05% from a 5% capric acid solution in ethanol andincubated at 30° C., with shaking at about 300 rpm. After 24 hoursincubation cultures were fed glycerol to 30 g/L and an additional bolusof 0.05% capric acid. Incubation continued before sampling for GC at 24,48, and 72 hours. The results are shown in FIG. 12. GC analysis showedthat a greater proportion of capric acid was converted to sebacic acidwhen particular elements of the omega-oxidation pathway are amplified.The data are presented as % of theoretical maximum yield. Strains whichinclude genetic modifications to CYPA18 and CYPA19 achieve approximately80% of theoretical maximum yield in conversion of capric acid to sebacicacid. The strain designated +CPR+A18 has about 30 copies of CYPA18,whereas the strain designated +CPR+A19 has about 7 copies of CYPA19.

Example 8: Conversion of Methyl-Laurate to Dodecanedioic Acid in ShakeFlask Fermentation

5 mL of SP92 glycerol medium (6.7 g/L yeast nitrogen base, 3.0 g/L yeastextract, 3.0 g/L (NH4)2SO4, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 75 g/Lglycerol) was inoculated with a single colony of a completelybeta-oxidation blocked strain of Candida tropicalis (ATCC20962), as wellas, modified derivatives of this strain with amplified components of theomega-oxidation pathway, and the cultures grown overnight at 30° C.,with shaking at about 250 rpm. The starter cultures were then used toinoculate 25 mL cultures of the same medium and grown overnight at 30°C., with shaking at about 250 rpm. Cells were pelleted by centrifugationfor 10 minutes at 4° C. and 1,050×g and the supernatant discarded. Cellswere resuspended in 12.5 mL SP92 glycerol medium and transferred to asterile 250 mL glass baffled flask. Cultures were fed 2% (v/v) methyllaurate and incubated at 30° C., with shaking at about 300 rpm. After 24hours incubation, cultures were fed glycerol to 60 g/L and incubationcontinued before sampling for

GC at 48 hours. GC analysis showed that amplification of certaincomponents of the omega oxidation pathway allow for increased conversionto dodecanedioic acid (FIG. 13).

Example 9: Alteration of Acyl CoA Oxidase Substrate Specificity

The substrate specificity of the peroxisomal acyl-CoA oxidase enzymesPOX4 and POX5 have been shown to be involved in the control of thediacid product chain-length in fermentations of Candida tropicalis fed amixed chain-length fatty acid feedstock. Reduction or elimination ofPOX4 activity, POX5 activity or POX4 activity and POX5 activity, effectsthe carbon chain-length distribution of dicarboxylic acids produced inC. tropicalis. Acyl-CoA oxidase is the first enzyme in the cyclicbeta-oxidation pathway that shortens a substrate by two carbons eachcycle. Thus the acyl-CoA oxidase activity serves as the pathway entrypoint for substrates entering into the beta-oxidation pathway. Alteringthe substrate specificity an acyl-CoA oxidase activity such that it isnot active on substrate carbon chains shorter than a desired carbonchain length (e.g., C8, C10, C12, C14 and the like), can inhibitshortening of carbon chains below a chosen threshold, allowingaccumulation of a desired target chain length and product (e.g., C12,dodecanedioic acid).

The native acyl-CoA oxidase isozymes in C. tropicalis, Pox4p and Pox5phave different substrate specificities. The Pox4p isozyme has a broadsubstrate specificity while the Pox5p isozyme has a narrow substratespecificity. In strains that are Pox4⁻, Pox5⁺ the chain length of thediacid product is determined by the substrate specificity of the Pox5pisozyme and the main product is adipic acid.

To maximize production of desired diacid products of longer chainlengths (e.g., C12) in fermentations, genetically modified organismscontaining an acyl-CoA oxidase activity with a substrate chain-lengthspecificity appropriate for the chain-length of the desired diacidproduct can be engineered, in some embodiments. The source of theacyl-CoA oxidase activity or the method of engineering the acyl-CoAoxidase activity may vary. Non-limiting examples of organisms which canbe used to provide polynucleotide sequences suitable for use inengineering altered substrate specificity acyl-CoA oxidase activitiesinclude; plants (e.g., Arabidopsis, Cucurbita (e.g., pumpkin, squash),Oryza (e.g., rice)); animals (e.g., Bos (e.g., bovine), Cavia (e.g.,guinea pig), Mus (e.g., mouse), Rattus (e.g., rat), Phascolarctos (e.g.,Koala), primates (e.g., orangutans)); molds (e.g., Dictyostelium (e.g.,slime molds)); insects (e.g., Drosophila); Yeast (e.g., Yarrowialipolyitica, Candida maltosa, Candida glabrata, Ashbya gossypii,Debaryomyces hansenii, Kluyveromyces lactis, Pichia pastoris,Saccharomyces cerevisiae); bacteria (e.g., Eschericia coli);cyanobacteria; nematodes (e.g., Caenorhabditis); and humans.

Acyl-CoA oxidase activities with different substrate chain-lengthspecificities can be identified by:

-   -   1) Selecting acyl-CoA oxidase genes from heterologous organisms        that contain different substrate chain-length specificities. The        identified genes can be transferred into a Candida strain        deleted for all acyl-CoA oxidase activity. The only acyl-CoA        oxidase activity detectable in such a genetically modified        organism may be that imparted by the heterologous gene.    -   2) Engineering an acyl-CoA oxidase gene library by domain        swapping from multiple acyl-CoA oxidase genes to produce a        library of non-native chimeric acyl-CoA oxidase genes. The        library of chimeric genes can be transferred into a strain of C.        tropicalis deleted for all acyl-CoA oxidase activity. The only        detectable acyl-CoA oxidase activity may be that imparted by an        engineered gene from the library of non-native chimeric acyl-CoA        oxidase genes.    -   3) Engineering an acyl-CoA oxidase gene library by random        mutagenesis. A naturally occurring or engineered acyl-CoA        oxidase activity with a substrate chain-length specificity close        to that desired can be used as the basis for random mutagenesis,        followed by screening and/or selection in an effort to generate        and identify an altered activity with the desired substrate        chain-length specificity. The library of genes can be        transferred into a Candida strain deleted for all acyl-CoA        oxidase activity. The only detectable acyl-CoA oxidase activity        may be that imparted by the gene from the randomly mutagenized        library.    -   4) Engineering an acyl-CoA oxidase gene by intelligent design        and directed mutation using protein structural information to        guide the position and identity of the amino acid(s) to be        replaced. The engineered gene(s) can be transferred into a        Candida strain deleted for all acyl-CoA oxidase activity. The        only detectable acyl-CoA oxidase activity may be that imparted        by the engineered gene(s).

A non-limiting example of a post-engineering method for selecting genesthat impart the desired substrate chain-length specificity is providedherein. Selection is performed by growth on substrates of differentchain lengths that are provided as the only carbon source. Growth of thecells on certain substrates but not others often reflects the substratechain-length specificity of the acyl-CoA oxidase enzyme present in thestrain. Candida tropicalis can utilize alkanes provided in the gas phaseas its sole carbon source for growth. Alkanes of different chain lengthsare provided by soaking a filter paper in the appropriate alkane, andinverting a solid growth media without a carbon source over the filterpaper, with each specific carbon source (e.g., specific chain lengthalkane) provided in a different petri dish. Serially diluted C.tropicalis carrying the altered specificity acyl-CoA oxidase genes arespotted on the solid growth media as a growth selection for thechain-length specificity of the acyl-CoA oxidase enzyme in each strain.Shown in FIGS. 14 and 15 are a schematic representation of the selectionprocess, which provides an alkane as a gas phase carbon source, asdescribed herein. The solid growth media is an agar medium containingyeast nitrogen base without amino acids or any other carbon source. Theplated cells are inverted over a lid containing a filter paper soakedwith an alkane of appropriate chain length that evaporates and providesthe carbon source through the gas phase, as shown in FIG. 14.

Candida strains containing altered acyl-CoA oxidase activities generatedas described herein are selected and/or screened using the methoddescribed herein. Strains carrying different altered acyl-CoA oxidaseactivities (e.g., strain 1 (S1), strain 2 (S2), strain 3 (S3), strain 4(S4)) are grown overnight in a rich medium (e.g., YPD). Overnightcultures are centrifuged and washed to remove any traces of residualrich medium and serial dilutions of the cells are prepared in aphosphate buffered solution. The serial dilutions of each strain arespotted onto multiple YNB agar plates (growth medium having no aminoacids or other carbon sources), the individual plates inverted overfilter papers soaked in the appropriate chain length alkane, and theplate incubated at 30° C. The growth of the strains is dependent uponthe chain-length specificity of the acyl-CoA oxidase. In order toutilize the particular alkane for growth the provided chain-length mustbe able to enter the beta-oxidation pathway. The shortest chain-lengthat which a certain strain is able to grow indicates the shortestchain-length of the acyl-CoA oxidase isozymes substrate specificity. Anexample is provided in FIG. 15. FIG. 15 illustrates that strain S4 cangrow on decane, but is unable to grow on octane. Therefore the modifiedacyl-CoA oxidase activity of strain S4 has a substrate chain-lengthspecificity that inhibits the utilization of 8 carbon molecules and thediacid product from fermentations with this strain typically result inan 8 carbon diacid. Acyl-CoA oxidase activities with any desiredspecificity can be selected and/or screened using the method describedherein.

It will be understood that the example presented herein is a generalizedmethod used to describe the selection/screening process. The feedstocksused for the selection and screening process are altered to suit theacyl-CoA oxidase activity being sought. For example, for acyl-CoAoxidases having specificity for longer chain substrates, feedstockshaving longer carbon chain lengths could be substituted to allowselection and or screening for acyl-CoA oxidase activities withspecificities for longer carbon chain lengths.

Example 10: Transformation of C. tropicalis Procedure

5 mL YPD start cultures were inoculated with a single colony of C.tropicalis and incubated overnight at 30° C., with shaking at about 200rpm. The following day, fresh 25 mL YPD cultures, containing 0.05%Antifoam B, were inoculated to an initial OD_(600nm) of 0.4 and theculture incubated at 30° C., with shaking at about 200 rpm until anOD_(600nm) of 1.0-2.0 was reached. Cells were pelleted by centrifugationat 1,000×g, 4° C. for 10 minutes. Cells were washed by resuspending in10 mL sterile water, pelleted, resuspended in 1 mL sterile water andtransferred to a 1.5 mL microcentrifuge tube. The cells were then washedin 1 mL sterile TE/LiOAC solution, pH 7.5, pelleted, resuspended in 0.25mL TE/LiOAC solution and incubated with shaking at 30° C. for 30minutes.

The cell solution was divided into 50 uL aliquots in 1.5 mL tubes towhich was added 5-8 ug of linearized DNA and 5 uL of carrier DNA (boiledand cooled salmon sperm DNA, 10 mg/mL). 300 uL of sterile PEG solution(40% PEG 3500, 1×TE, 1×LiOAC) was added, mixed thoroughly and incubatedat 30° C. for 60 minutes with gentle mixing every 15 minutes. 40 uL ofDMSO was added, mixed thoroughly and the cell solution was incubated at42° C. for 15 minutes. Cells were then pelleted by centrifugation at1,000×g 30 seconds, resuspended in 500 uL of YPD media and incubated at30° C. with shaking at about 200 rpm for 2 hours. Cells were thenpelleted by centrifugation and resuspended in 1 mL 1×TE, cells werepelleted again, resuspended in 0.2 mL 1×TE and plated on selectivemedia. Plates were incubated at 30° C. for growth of transformants.

Example 11: Procedure for Recycling of the URA3 Marker

The URA3 gene was obtained from genomic DNA of Candida yeast cultureATCC20336. C. tropicalis has a limited number of selectable marker, ascompared to S. cerevisiae, therefore, the URA3 marker is “recycled” toallow multiple rounds of selection using URA3. To reutilize the URA3marker for subsequent engineering of C. tropicalis, a single colonyhaving the Ura⁺ phenotype was inoculated into 3 mL YPD and grownovernight at 30° C. with shaking. The overnight culture was thenharvested by centrifugation and resuspended in 1 mL YNB+YE (6.7 g/LYeast Nitrogen Broth, 3 g/L Yeast Extract). The resuspended cells werethen serially diluted in YNB+YE and 100 uL aliquots plated on YPD plates(incubation overnight at 30° C.) to determine titer of the originalsuspension. Additionally, triplicate 100 uL aliquots of the undilutedsuspension were plated on SC Dextrose (Bacto Agar 20 g/L, Uracil 0.3g/L, Dextrose 20 g/L, Yeast Nitrogen Broth 6.7 g/L, Amino Acid DropoutMix 2.14 g/L) and 5-FOA.at 3 different concentrations (0.5, 0.75, 1mg/mL).

Plates were incubated for at least 5 days at 30° C. Colonies arising onthe SC Dextrose+5-FOA plates were resuspended in 50 uL sterile,distilled water and 5 uL utilized to streak on to YPD and SC-URA (SCDextrose medium without Uracil) plates. Colonies growing only on YPD andnot on SC-URA plates were then inoculated into 3 mL YPD and grownovernight at 30° C. with shaking. Overnight cultures were harvested bycentrifugation and resuspended in 1.5 mL YNB (6.7 g/L Yeast NitrogenBroth). The resuspended cells were serially diluted in YNB and 100 uLaliquots plated on YPD plates and incubation overnight at 30° C. todetermine initial titer. 1 mL of each undiluted cell suspension also wasplated on SC-URA and incubated for up to 7 days at 30° C. Colonies onthe SC-URA plates are revertants and the isolate with the lowestreversion frequency (<10⁻⁷) was used for subsequent strain engineering.

Example 12: Cloning and Analysis of C. tropicalis Fatty Alcohol Oxidase(FAO) Alleles

Isolation of Fatty Alcohol Oxidase Genes from C. tropicalis

C. tropicalis (ATCC20336) fatty alcohol oxidase genes were isolated byPCR amplification using primers generated to amplify the sequence regioncovering promoter, fatty alcohol oxidase gene (FAO) and terminator ofthe FAO1 sequence (GenBank accession number of FAO1 AY538780). Theprimers used to amplify the fatty alcohol oxidase nucleotide sequencesfrom Candida strain ATCC20336, are showing in the table below.

Oligonucleotides for cloning FAO alleles Oligo Sequence oAA0144AACGACAAGATTAGATTGGTTGAGA oAA0145 GTCGAGTTTGAAGTGTGTGTCTAAG oAA0268AGATCTCATATGGCTCCATTTTTGCCCGACCAGGTCGA CTACAAACACGTC oAA0269ATCTGGATCCTCATTACTACAACTTGGCTTTGGTCTTC AAGGAGTCTGCCAAACCTAAC oAA0282ACATCTGGATCCTCATTACTACAACTTGGCCTTGGTCT oAA0421CACACAGCTCTTCTAGAATGGCTCCATTTTTGCCCGAC CAGGTCGAC oAA0422CACACAGCTCTTCCTTTCTACAACTTGGCTTTGGTCTT CAAGGAGTCTGC oAA0429GTCTACTGATTCCCCTTTGTC oAA0281 TTCTCGTTGTACCCGTCGCA

PCR reactions contained 25 uL 2× master mix, 1.5 uL of oAA0144 andoAA0145 (10 uM), 3.0 uL genomic DNA, and 19 uL sterile H₂O.Thermocycling parameters used were 98° C. for 2 minutes, 35 cycles of98° C. 20 seconds, 52° C. 20 seconds, 72° C. 1 minute, followed by 72°C. 5 minutes and a 4° C. hold. PCR products of the correct size were gelpurified, ligated into pCR-Blunt II-TOPO (Invitrogen) and transformedinto competent TOP10 E. coli cells (Invitrogen). Clones containing PCRinserts were sequenced to confirm correct DNA sequence. Four FAO alleleswere identified from sequence analysis and designated as FAO-13, FAO-17,FAO-18 and FAO-20. The sequence of the clone designated FAO-18 had asequence that was substantially identical to the sequence of FAO1 fromGenBank. The resulting plasmids of the four alleles were designatedpAA083, pAA084, pAA059 and pAA085, respectively. Sequence identitycomparisons of FAO genes isolated as described herein are shown in thetables below.

DNA sequence identity FAO- FAO- FAO- FAO- FAO1 18 17 13 20 FAO2a FAO2bFAO1 100 100 98 96 95 83 82 FAO-18 100 98 96 95 83 82 FAO-17 100 98 9883 82 FAO-13 100 99 83 83 FAO-20 100 83 83 FAO2a 100 96 FAO2b 100

Protein sequence identity FAO- FAO- FAO- FAO- FAO1 18 17 13 20 FAO2aFAO2b FAO1 100 100 99 98 98 81 80 FAO-18 100 99 98 98 81 80 FAO-17 10099 99 82 81 FAO-13 100 99 82 81 FAO-20 100 82 81 FAO2a 100 97 FAO2b 100

Amino acid differences in FAO alleles 32 75 89 179 185 213 226 352 544590 FAO1 E M G L Y T R H S P FAO-13 Q T A L Y A K Q A A FAO-20 Q T A M DA K Q A A

Expression of FAO Alleles in E. coli

To determine the levels of FAO enzyme activity with respect to variouscarbon sources, the four isolated FAO alleles were further cloned andover-expressed in E. coli. The FAOs were amplified using the plasmidsmentioned above as DNA template by PCR with primers oAA0268 and oAA0269for FAO-13 and FAO-20 and oAA0268 and oAA0282 for FAO-17 and FAO-18,using conditions as described herein. PCR products of the correct sizewere gel purified and ligated into pET11a vector between NdeI and BamHIsites and transformed into BL21 (DE3) E. coli cells. The coloniescontaining corresponding FAOs were confirmed by DNA sequencing.Unmodified pET11a vector also was transformed into BL21 (DE3) cells, asa control. The resulting strains and plasmids were designated sAA153(pET11a), sAA154 (pAA079 containing FAO-13), sAA155 (pAA080 containingFAO-17), sAA156 (pAA081 containing FAO-18) and sAA157 (pAA082 containingFAO-20), respectively. The strains and plasmids were used for FAOover-expression in E. coli. One colony of each strain was transferredinto 5 mL of LB medium containing 100 μg/mL ampicillin and grownovernight at 37° C., 200 rpm. The overnight culture was used toinoculate a new culture to OD_(600nm) 0.2 in 25 ml LB containing 100μg/ml ampicillin. Cells were induced at OD_(600nm) 0.8 with 0.3 mM IPTGfor 3 hours and harvested by centrifugation at 4° C. 1,050×g for 10minutes. The cell pellet was stored at −20° C.

Expression of FAOs in C. tropicalis

Two alleles, FAO-13 and FAO-20, were chosen for amplification in C.tropicalis based on their substrate specificity profile, as determinedfrom enzyme assays of soluble cell extracts of E. coli with overexpressed FAOs. DNA fragments containing FAO-13 and FAO-20 wereamplified using plasmids pAA079 and pAA082 as DNA templates,respectively, by PCR with primers oAA0421 and oAA0422. PCR products ofthe correct sizes were gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing FAO inserts were sequenced to confirmcorrect DNA sequence. Plasmids containing FAO-13 and FAO-20 weredigested with SapI and ligated into vector pAA105, which includes the C.tropicalis PGK promoter and terminator. The resulting plasmids wereconfirmed by restriction digestion and DNA sequencing and designated aspAA115 (FAO-13) and pAA116 (FAO-20), respectively. Plasmids pAA115 andpAA116 were linearized with SpeI, transformed into competent C.tropicalis Ura⁻ strains sAA002 (SU-2, ATCC20913) and sAA103. Theintegration of FAO-13 and FAO-20 was confirmed by colony PCR usingprimers oAA0429 and oAA0281. The resulting strains were designated assAA278 (pAA115 integrated in strain sAA002), sAA280 (pAA116 integratedin sAA002), sAA282 (pAA115 integrated in sAA103), and sAA284 (pAA116integrated in sAA103), and were used for fatty alcohol oxidaseover-expression in C. tropicalis.

One colony of each strain was inoculated into 5 ml YPD and grownovernight as described herein. The overnight culture was used toinoculate a new 25 mL YPD culture to about OD_(600nm) 0.5. FAOover-expression was regulated by the PGK promoter/terminator, inducedwith glucose in the medium and expressed constitutively. Strains sAA002and sAA103 (e.g., untransformed starting strains) were included asnegative controls for FAO over-expression. Cells were harvested at earlylog phase (OD_(600nm)=in the range of between about 3 to about 5) bycentrifugation at 4° C. for 10 minutes at 1,050× g. Cell pellets werestored at −20° C.

Cell Extract Preparation from E. coli

Cell pellets from 25 mL of FAO expressing E. coli cultures wereresuspended in 10 mL phosphate-glycerol buffer containing 50 mMpotassium phosphate buffer (pH7.6), 20% glycerol, 1 mMPhenylmethylsulfonyl fluoride (PMSF), 2 uL Benzonase 25 U/uL, 20 uL 10mg/mL lysozyme. The cells were then lysed by incubation at roomtemperature for 50 minutes on a rotating shaker, and the cell suspensioncentrifuged for 30 minutes at 4′C using 15,000×g for. The supernatantwas aliquoted in 1.5 ml microcentrifuge tubes and stored at −20° C. forFAO enzyme activity assays.

Cell Extract Preparation from C. tropicalis

Frozen C. tropicalis cell pellets were resuspended in 1.2 ml ofphosphate-glycerol buffer containing 50 mM potassium phosphate buffer(pH7.6), 20% glycerol, 1 mM Phenylmethylsulfonyl fluoride (PMSF).Resuspended cells were transferred to 1.5 mL screw-cap tubes containingabout 500 uL of zirconia beads on ice. The cells were lysed with a BeadBeater (Biospec) using 2 minute pulses and 1 minute rest intervals onice. The process was repeated 3 times. The whole cell extract was thentransferred to a new 1.5 ml tube and centrifuged at 16,000×g for 15minutes at 4° C. The supernatant was transferred into a new tube andused for FAO enzyme activity assays.

Protein Concentration Determination

Protein concentration of the cell extracts was determined using theBradford Reagent following manufacturers' recommendations (Cat#23238,Thermo Scientific).

FAO Enzyme Activity Assay

FAO enzyme activity assays were performed using a modification of Eirichet al., 2004). The assay utilizes a two-enzyme coupled reaction (e.g.,FAO and horse radish peroxidase (HRP)) and can be monitored byspectrophotometry. 1-Dodecanol was used as a standard substrate forfatty alcohol oxidase enzymatic activity assays. FAO oxidizes thedodecanol to dodecanal while reducing molecular oxygen to hydrogenperoxide simultaneously. HRP reduces (2,2′-azino-bis3-ethylbenzthiazoline-6-sulfonic acid; ABTS) in the two-enzyme coupledreaction, where the electron obtained from oxidizing hydrogen peroxideto ABTS, which can be measured by spectrometry at 405 nm. The assay wasmodified using aminotriazole (AT) to prevent the destruction of H₂O₂ byendogenous catalase, thus eliminating the need for microsomalfractionation. The final reaction mixture (1.0 mL) for FAO enzyme assayconsisted of 500 μL of 200 mM HEPES buffer, pH 7.6; 50 μL of a 10 mg/mLABTS solution in deionized water; 10 μL of 5 mM solution of dodecanol inacetone; 40 μL of 1M AT and 5 μL of a 2 mg/mL horseradish peroxidasesolution in 50 mM potassium phosphate buffer, pH 7.6. Reaction activitywas measured by measuring light absorbance at 405 nm for 10 minutes atroom temperature after adding the extract. The amount of extract addedto the reaction mixture was varied so that the activity fell within therange of 0.2 to 1.0 ΔA_(405nm)/min. The actual amounts of extract usedwere about 1.69 U/mg for E. coli expressed FAO-13, 0.018 U/mg for E.coli expressed FAO-17, 0.35 U/mg for E. coli expressed FAO-18 (e.g.,FAO1), 0.47 U/mg E. coli expressed FAO-20, 0.036 U/mg C. tropicalis(strain sAA278) expressed FAO-13, 0.016 U/mg C. tropicalis (strainsAA282) expressed FAO-13, 0.032 U/mg C. tropicalis (strain sAA280)expressed FAO-20 and 0.029 U/mg C. tropicalis (strain sAA284) expressedFAO-20. FAO activity was reported as activity units/mg of total protein(1 unit=1 μmole substrate oxidized/min). An extinction coefficient at405 nm of 18.4 was used for ABTS and was equivalent to 0.5 mM oxidizedsubstrate. The results of the activity assays are shown in the tablesbelow.

FAO activity (units/mg total protein) on primary alcohols 1- 1- 1- 1- 1-1- 1- Butanol Pentanol Hexanol Octanol Decanol Dodecanol TetradecanolHexadecanol FAO-13 0.01 0.09 1.17 82.67 70.94 100 79.35 58.88 FAO-170.72 0.26 1.06 66.23 22.00 100 47.86 60.98 FAO-18 0.07 0.11 0.26 60.5654.56 100 114.47 50.65 FAO-20 0.07 0.11 0.91 55.96 74.57 100 89.52 42.59FAO activity (units/mg total protein) on omega hydroxy fatty acids 1-Dodecanol 6-OH-HA 10-OH-DA 12-OH-DDA 16-OH-HDA FAO-13 100 4.18 4.14 6.878.57 FAO-17 100 1.18 0.00 0.59 0.94 FAO-18 100 0.00 0.00 4.87 2.94FAO-20 100 0.03 0.04 2.25 7.46

Example 13: Construction of C. tropicalis Shuttle Vector pAA061

Vector pAA061 was constructed from a pUC19 backbone to harbor theselectable marker URA3 from Candida strain ATCC20336 as well asmodifications to allow insertion of C. tropicalis promoters andterminators. A 1,507 bp DNA fragment containing the promoter, ORF, andterminator of URA3 from C. tropicalis ATCC20336 was amplified usingprimers oAA0124 and oAA0125, shown in the table below. The URA3 PCRproduct was digested with NdeI/MluI and ligated into the 2,505 bpfragment of pUC19 digested with NdeI/BsmBI (an MluI compatible overhangwas produced by BsmBI). In order to replace the lac promoter with ashort 21 bp linker sequence, the resulting plasmid was digested withSphI/SapI and filled in with a linker produced by annealing oligosoAA0173 and oAA0174. The resulting plasmid was designated pAA061.

Oligonucleotides for construction of pAA061 PCR product Oligos Sequence(bp) oAA0124 cacacacatatgCGACGGGTACAACGAGAATT 1507 oAA0125cacacaacgcgtAGACGAAGCCGTTCTTCAAG oAA0173 ATGATCTGCCATGCCGAACTC   21oAA0174 AGCGAGTTCGGCATGGCAGATCATCATG (linker)

Example 14: Cloning of C. Tropicalis PGK Promoter and Terminator

Vector pAA105 was constructed from base vector pAA061 to include thephosphoglycerate kinase (PGK) promoter and terminator regions from C.tropicalis ATCC20336 with an intervening multiple cloning site (MCS) forinsertion of open reading frames (ORF's). The PGK promoter region wasamplified by PCR using primers oAA0347 and oAA0348, shown in the tablebelow. The 1,029 bp DNA fragment containing the PGK promoter wasdigested with restriction enzymes PstI/XmaI. The PGK terminator regionwas amplified by PCR using primers oAA0351 and oAA0352, also shown inthe table below. The 396 bp DNA fragment containing the PGK terminatorwas digested with restriction enzymes XmaI/EcoRI. The 3,728 bpPstI/EcoRI DNA fragment from pAA061 was used in a three piece ligationreaction with the PGK promoter and terminator regions to produce pAA105.The sequence between the PGK promoter and terminator containsrestriction sites for incorporating ORF's to be controlled by thefunctionally linked constitutive PGK promoter.

Oligonucleotides for cloning C. tropicalis PGK promoter and terminatorPCR product Oligos Sequence (bp) oAA0347CACACACTGCAGTTGTCCAATGTAATAATTTT 1028 oAA0348CACACATCTAGACCCGGGCTCTTCTTCTGAAT AGGCAATTGATAAACTTACTTATC oAA0351GAGCCCGGGTCTAGATGTGTGCTCTTCCAAAG 396 TACGGTGTTGTTGACA oAA0352CACACACATATGAATTCTGTACTGGTAGAGCT AAATT

Example 15: Cloning of the POX4 Locus

Primers oAA0138 and oAA0141 (shown in the table below) were generated toamplify the entire sequence of NCBI accession number M12160 for theYSAPOX4 locus from genomic DNA prepared from Candida strain ATCC20336.The 2,845 bp PCR product was cloned into the vector, pCR-BluntII-TOPO(Invitrogen), sequenced and designated pAA052.

Oligonucleotides for cloning of POX4 Oligos Sequence PCR product (bp)oAA0138 GAGCTCCAATTGTAATATTTCGGG 2845 oAA0141 GTCGACCTAAATTCGCAACTATCAA

Example 16: Cloning of the POX5 Locus

Primers oAA0179 and oAA0182 (shown in the table below) were generated toamplify the entire sequence of NCBI accession number M12161 for theYSAPOX5 locus from genomic DNA prepared from Candida strain ATCC20336.The 2,624 bp PCR product was cloned into the vector, pCR-BluntII-TOPO(Invitrogen), sequenced and designated pAA049.

Oligonucleotides for cloning of POX5 PCR product Oligos Sequence (bp)oAA0179 GAATTCACATGGCTAATTTGGCCTCGG 2624 TTCCACAACGCACTCAGCATTAAAAAoAA0182 GAGCTCCCCTGCAAACAGGGAAACACT TGTCATCTGATTT

Example 17: Construction of Strains with Amplified CPR and CYP52 Genes

Strains having an increased number of copies of cytochrome P450reductase (CPR) and/or for cytochrome P450 monooxygenase (CYP52) geneswere constructed to determine how over expression of CPR and CYP52affected diacid production.

Cloning and Integration of the CPR Gene.

A 3,019 bp DNA fragment encoding the CPR promoter, ORF, and terminatorfrom C. tropicalis ATCC750 was amplified by PCR using primers oAA0171and oAA0172 (see table below) incorporating unique SapI and SphI sites.The amplified DNA fragment was cut with the indicated restrictionenzymes and ligated into plasmid pAA061, (described in Example 13) toproduce plasmid pAA067. Plasmid pAA067 was linearized with ClaI andtransformed into C. tropicalis Ura⁻ strain sAA103 (ura3/ura3,pox4::ura3/pox4::ura3, pox5::ura3/pox5::ura3). Transformations wereperformed with plasmid pAA067 alone and in combination with plasmidsharboring the CYP52A15 or CYP52A16 genes, described below.

Cloning and Integration of CYP52A15 Gene.

A 2,842 bp DNA fragment encoding the CYP52A15 promoter, ORF, andterminator from C. tropicalis ATCC20336 was amplified by PCR usingprimers oAA0175 and oAA0178 (see table below) and cloned intopCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A15 DNAfragment was isolated by restriction digest with XbaI/BamHI (2,742 bp)and ligated into plasmid pAA061, (described in Example 13), to produceplasmid pAA077. Plasmid pAA077 was linearized with PmlI and transformedinto C. tropicalis Ura⁻ strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3,pox5::ura3/pox5::ura3). pAA077 was cotransformed with plasmid pAA067harboring the CPR gene.

Cloning and Integration of CYP52A16 Gene.

A 2,728 bp DNA fragment encoding the CYP52A16 promoter, ORF, andterminator from C. tropicalis ATCC20336 was amplified by PCR usingprimers oAA0177 and oAA0178 (see table below) and cloned intopCR-BluntII-TOPO for DNA sequence verification. The cloned CYP52A16 DNAfragment was amplified with primers oAA0260 and oAA0261 (see tablebelow) which incorporated unique SacI/XbaI restriction sites. Theamplified DNA fragment was digested with SacI and XbaI restrictionenzymes and ligated into plasmid pAA061 to produce plasmid pAA078.Plasmid pAA078 was linearized with ClaI and transformed into C.tropicalis Ura⁻ strain sAA103 (ura3/ura3, pox4::ura3/pox4::ura3,pox5::ura3/pox5::ura3). pAA078 was cotransformed with plasmid pAA067harboring the CPR gene.

Oligonucleotides for cloning of CPR, CYP52A15 and CYP52A16 PCR productOligos Sequence (bp) oAA0171 cacctcgctcttccAGCTGTCATGTCTAT 3019TCAATGCTTCGA oAA0172 cacacagcatgcTAATGTTTATATCGTTG ACGGTGAAA oAA0175cacaaagcggaagagcAAATTTTGTATTC 2842 TCAGTAGGATTTCATC oAA0178cacacagcatgCAAACTTAAGGGTGTTGT AGATATCCC oAA0177cacacacccgggATCGACAGTCGATTACG 2772 TAATCCATATTATTT oAA0178cacacagcatgCAAACTTAAGGGTGTTGT AGATATCCC oAA0260cacacagagctcACAGTCGATTACGTAATCCAT 2772 oAA0261cacatctagaGCATGCAAACTTAAGGGTGTTGTA

Preparation of Genomic DNA.

Genomic DNA was prepared from transformants for PCR verification and forSouthern blot analysis. Isolated colonies were inoculated into 3 mL YPDand grown overnight at 30° C. with shaking. Cells were pelleted bycentrifugation. To each pellet, 200 uL Breaking Buffer (2% Triton X-100,1% SDS, 100 mM NaCl, 10 mM Tris pH 8 and, 1 mM EDTA) was added, and thepellet resuspended and transferred to a fresh tube containing 200 uL 0.5mm Zirconia/Silica Beads. 200 uL Phenol:Chloroform:Isoamyl Alcohol(25:24:1) was added to each tube, followed by vortexing for 1 minute.Sterile distilled water was added (200 uL) to each tube and the tubeswere centrifuged at 13000 rpm for 10 minutes. The aqueous layer wasethanol precipitated and washed with 70% ethanol. The pellet wasresuspended in 100-200 μl 10 mM Tris, after drying. Genomic DNApreparation for southern blot analysis was performed using the sameprocedure on 25 mL cultures for each colony tested.

Characterization of Strains with Amplified CPR and CYP52 Genes.

Verification of integrants was performed by PCR using primers oAA0252and oAA0256 (CPR), oAA0231 and oAA0281 (CYP52A15), and oAA242 andoAA0257 (CYP52A16). The primers used for verification are shown in thetable below.

Oligonucleotides for PCR verification of CPR, CYP52A15 and CYP52A16Oligos Sequence PCR product (bp) oAA0252 TTAATGCCTTCTCAAGACAA 743oAA0256 GGTTTTCCCAGTCACGACGT oAA0231 CCTTGCTAATTTTCTTCTGTATAGC 584oAA0281 TTCTCGTTGTACCCGTCGCA oAA0242 CACACAACTTCAGAGTTGCC 974 oAA0257TCGCCACCTCTGACTTGAGC

Southern blot analysis was used to determine the copy number of the CPR,CYP52A15 and CYP52A16 genes. Biotinylated DNA probes were prepared withgene specific oligonucleotides using the NEBlot Phototope Kit from NewEngland BioLabs (Catalog #N7550S) on PCR products generated from eachgene target as specified in the table below. Southern Hybridizationswere performed using standard methods (e.g., Sambrook, J. and Russell,D. W. (2001) Molecular Cloning: A Laboratory Manual, (3^(rd) ed.), pp.6.33-6.64. Cold Spring Harbor Laboratory Press). Detection of hybridizedprobe was performed using the Phototope-Star Detection Kit from NewEngland BioLabs (Catalog #N7020S). Copy number was determined bydensitometry of the resulting bands.

Oligonucleotides for Probe Template PCR of CPR, CYP52A15 and CYP52A16Oligos Sequence Gene Template PCR product (bp) oAA0250AATTGAACATCAGAAGAGGA CPR pAA067 1313 oAA0254 CCTGAAATTTCCAAATGGTGTCTAAoAA0227 TTTTTTGTGCGCAAGTACAC CYP52A15 pAA077 905 oAA0235CAACTTGACGTGAGAAACCT oAA0239 AGATGCTCGTTTTACACCCT CYP52A16 pAA078 672oAA0247 ACACAGCTTTGATGTTCTCT

Example 18: Addition and/or Amplification of Monooxygenase andMonooxygenase Reductase Activities

Cytochrome P450's often catalyze a monooxygenase reaction, e.g.,insertion of one atom of oxygen into an organic substrate (RH) while theother oxygen atom is reduced to water:

RH+O2+2H++2e-→ROH+H2O

The substrates sometimes are of a homogeneous carbon chain length.Enzymes with monooxygenase activity sometimes recognize substrates ofspecific carbon chain lengths, or a subgroup of carbon chain lengthswith respect to organic substrates of homogenous carbon chain length.Addition of novel cytochrome activities (e.g., B. megaterium BM3) and/oramplification of certain or all endogenous or heterologous monooxygenaseactivities (e.g., CYP52A12 polynucleotide, CYP52A13 polynucleotide,CYP52A14 polynucleotide, CYP52A15 polynucleotide, CYP52A16polynucleotide, CYP52A17 polynucleotide, CYP52A18 polynucleotide,CYP52A19 polynucleotide, CYP52A20 polynucleotide, CYP52D2polynucleotide, BM3 polynucleotide) can contribute to an overallincrease in carbon flux through native and/or engineered metabolicpathways, in some embodiments. In certain embodiments, adding a novelmonooxygenase or increasing certain or all endogenous or heterologousmonooxygenase activities can increase the flux of substrates of specificcarbon chain length or subgroups of substrates with mixtures of specificcarbon chain lengths. In some embodiments, the selection of amonooxygenase activity for amplification in an engineered strain isrelated to the feedstock utilized for growth of the engineered strain,pathways for metabolism of the chosen feedstock and the desire endproduct (e.g., dodecanedioic acid).

Strains engineered to utilize plant-based oils for conversion tododecanedioic acid can benefit by having one or more monooxygenaseactivities with substrate specificity that matches the fatty acidchain-length distribution of the oil. For example, the most prevalentfatty acid in coconut oil is lauric acid (12 carbons long), therefore,the monooxygenase activity chosen for a coconut oil-utilizing strain canhave a substrate preference for C12 fatty acids. For strains engineeredto utilize other plant based oils with different fatty acid chain-lengthdistributions it may be desirable to amplify a monooxygenase activitythat has a matching substrate preference. In some embodiments, a geneticmodification that alters monooxygenase activity increases the activityof one or more monooxygenase activities with a substrate preference forfeedstocks having carbon chain lengths of between about 12 and about 24carbons (e.g., mixed chain length alkanes, mixed chain length fattyacids, soapstocks, the like and combinations thereof). In certainembodiments, the genetic modification increases the activity of amonooxygenase activity with a preference for fatty acids having a carbonchain-length distribution of between about 10 carbons and about 16carbons.

As mentioned previously, the enzymes that carry out the monooxygenaseactivity are reduced by the activity of monooxygenase reductase, therebyregenerating the enzyme. Selection of a CPR for amplification in anengineered strain depends upon which P450 is amplified, in someembodiments. A particular CPR may interact preferentially with one ormore monooxygenase activities, in some embodiments, but not well withother monooxygenases. A monooxygenase reductase from Candida strainATCC750, two monooxygenase reductase activities from Candida strainATCC20336 and a monooxygenase reductase activity from Bacillusmegaterium are being evaluated for activity with the added and/oramplified monooxygenases described herein. Provided in the tables beloware nucleotide sequences used to add or amplify monooxygenase andmonooxygenase reductase activities.

Example 19: Amplification of Selected Beta Oxidation Activities

Described herein are methods of amplifying a POX5 beta oxidationactivity. Substantially similar methods can be utilized to amplifydifferent beta oxidation activities including non-POX (e.g., acyl-CoAoxidase) activities and/or acyl-CoA oxidase activities with alteredsubstrate specificities, as described herein.

Construction of POX5 Amplified Strains

Plasmid pAA166 (P_(POX4)POX5T_(POX4))

A PCR product containing the nucleotide sequence of POX5 was amplifiedfrom C. tropicalis 20336 genomic DNA using primers oAA540 and oAA541.The PCR product was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence. One such plasmid was designated, pAA165. PlasmidpAA165 was digested with BspQI and a 2-kb fragment was isolated. PlasmidpAA073 which contained a POX4 promoter and POX4 terminator was alsodigested with BspQI and gel purified. The isolated fragments wereligated together to generate plasmid pAA166. Plasmid pAA166 contains aP_(POX4)POX5T_(POX4) fragment.

Plasmid pAA204 (Thiolase Deletion Construct)

A PCR product containing the nucleotide sequence of a short-chainthiolase (e.g., acetyl-coA acetyltransferase) was amplified from C.tropicalis 20336 genomic DNA using primers oAA640 and oAA641. The PCRproduct was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence. One such plasmid was designated, pAA184. A URA3PCR product was amplified from pAA061 using primers oAA660 and oAA661.The PCR product was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed as described and clones containing PCR insertswere sequenced to confirm the correct DNA sequence. One such plasmid wasdesignated pAA192. Plasmid pAA184 was digested with BglII/SalI and gelpurified. Plasmid pAA192 was digested with BglII/SalI and a 1.5 kbfragment was gel purified. The isolate fragments were ligated togetherto create pAA199. An alternative P_(URA3) PCR product was amplified fromplasmid pAA061 using primers oAA684 and oAA685. The PCR product was gelpurified and ligated into pCR-BluntII-TOPO (Invitrogen), transformed asdescribed and clones containing PCR inserts were sequenced. One suchplasmid was designated, pAA201. Plasmid pAA199 was digested with SalIand gel purified. Plasmid pAA201 was digested with SalI and a 0.43 kbP_(URA3) was gel purified. The isolated fragments were ligated to createplasmid pAA204 that contains a direct repeat of P_(URA3).

Plasmid pAA221 (P_(POX4)POX5T_(POX4) in thiolase deletion construct)

A PCR product containing the nucleotide sequence of P_(POX4)POX5T_(POX4)was amplified from plasmid pAA166 DNA using primers oAA728 and oAA729.The PCR product was gel purified and ligated into pCR-BluntII-TOPO,transformed as described and clones containing PCR inserts weresequenced to confirm the sequence of the insert. One such plasmid wasdesignated, pAA220. Plasmid pAA204 was digested with BglII, treated withshrimp alkaline phosphatase (SAP), and a 6.5 kb fragment was gelpurified. Plasmid pAA220 was digested with BglII and a 2.7 kb fragmentcontaining P_(POX4)POX5T_(POX4) was gel purified. The isolated fragmentswere ligated to create plasmid pAA221.

Strain sAA617 (P_(POX4)POX5T_(POX4) in sAA451)

Strain sAA451 is a ura-, partially β-oxidation blocked Candida strain(ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5). Plasmid pAA221 wasdigested with EcoRI to release a DNA fragment containingP_(POX4)POX⁵T_(POX4) in a thiolase deletion construct. The DNA wascolumn purified and transformed to strain sAA451 to plate on SCD-uraplate. After two days, colonies were streaked out on YPD plates, singlecolonies selected and again streaked out on YPD plates. Single colonieswere selected from the second YPD plates and characterized by colonyPCR. The insertion of P_(POX4)POX5T_(POX4) in strain sAA451, disruptingthe short-chain thiolase gene, was confirmed by PCR and one such strainwas designated sAA617.

Strain sAA620

Strain sAA617 was grown overnight on YPD medium and plated onSCD+URA+5-FOA, to select for loop-out of URA3. Colonies were streakedout onto YPD plates twice as described for strain sAA617, and singlecolonies characterized by colony PCR. The loop-out of URA3 by directrepeats of PURA3 was confirmed by PCR. One such strain was designatedsAA620. Strain sAA620 has one additional copy of POX5 under control ofthe POX4 promoter.

Plasmid pAA156

A PCR product containing the nucleotide sequence of CYP52A19 wasamplified from Candida strain 20336 genomic DNA, using primers oAA525and oAA526. The PCR product was gel purified and ligated into pCR-BluntII-TOPO, transformed as described, and clones containing PCR insertswere sequenced to confirm correct DNA sequence. One such plasmid wasdesignated, pAA144. Plasmid pAA144 was digested with BspQI and a 1.7-kbfragment was isolated. Plasmid pAA073, which includes a POX4 promoterand POX4 terminator, also was digested with BspQI and gel purified. Theisolated fragments were ligated together to generate plasmid, pAA156.Plasmid pAA156 included P_(POX4)CYP52A19T_(POX4) fragment and URA3.

Strain sAA496

Plasmid pAA156 was digested with ClaI and column purified. Strain sAA451was transformed with this linearized DNA and plated on SCD-ura plate.Colonies were checked for CYP52A19 integration. Colonies positive forplasmid integration were further analyzed by qPCR to determine thenumber of copies of CYP52A19 integrated. One such strain, designatedsAA496 contained about 13 copies of the monooxygenase activity encodedby CYP52A19.

Strains sAA632 and sAA635

Strain sAA620 was transformed with linearized pAA156 DNA and plated onSCD-ura plates. Several colonies were checked for CYP52A19 integration.Colonies positive for plasmid integration were further analyzed by qPCRto determine the number of copies of CYP52A19 integrated. One suchstrain, designated sAA632 contained about 27 copies of the monooxygenaseactivity encoded by CYP52A19. Another strain, designated sAA635,contained about 12 copies of the monooxygenase activity encoded byCYP52A19.

Example 20: Cloning of C. tropicalis ACH Genes

ACH PCR product was amplified from C. tropicalis 20336 genomic DNA usingprimers oAA1095 and oAA1096, shown in the table below. The PCR productwas gel purified and ligated into pCR-BluntII-TOPO (Invitrogen),transformed into competent TOP10 E. coli cells (Invitrogen) and clonescontaining PCR inserts were sequenced to confirm correct DNA sequence.

Sequence analysis of multiple transformants revealed the presence ofallelic sequences for the ACH gene, which were designated ACHA and ACHB.A vector containing the DNA sequence for the ACHA allele was generatedand designated pAA310 (see FIG. 51). A vector containing the DNAsequence for the ACHB allele was generated and designated pAA311.

Primer sequence oAA1095 CACACACCCGGGATGATCAGAACCGTCCGTTATCAAT oAA1096CACACATCTAGACTCTCTTCTATTCTTAATTGCCGCT TCCACTAAACGGCAAAGTCTCCACG

Example 21: Cloning of C. tropicalis FAT1 Gene

FAT/PCR product was amplified from C. tropicalis 20336 genomic DNA usingprimers oAA1023 and oAA1024, shown in the table below. The PCR productwas gel purified and ligated into pCR-BluntII-TOPO (Invitrogen),transformed into competent TOP10 E. coli cells (Invitrogen) and clonescontaining PCR inserts were sequenced to confirm correct DNA sequence. Avector containing the DNA sequence for the FAT1 gene was designatedpAA296.

Primer sequence oAA1023 GATATTATTCCACCTTCCCTTCATT oAA1024CCGTTAAACAAAAATCAGTCTGTAAA

Example 22: Cloning of C. tropicalis ARE1 and ARE2 Genes

ARE1 and ARE2 PCR products were amplified from C. tropicalis 20336genomic DNA using primers oAA2006/oAA2007 and oAA1012/oAA1018,respectively, shown in the table below. The PCR products were gelpurified and ligated into pCR-Blunt II-TOPO (Invitrogen), transformedinto competent TOP10 E. coli cells (Invitrogen) and clones containingPCR inserts were sequenced to confirm correct DNA sequence. A vectorcontaining the DNA sequence for the ARE1 gene was designated pAA318. Avector containing the DNA sequence for the ARE2 gene was designatedpAA301.

Primer sequence oAA1012 ATGTCCGACGACGAGATAGCAGGAATAGTCAT oAA1018TCAGAAGAGTAAATACAACGCACTAACCAAGCT oAA2006 ATGCTGAAGAGAAAGAGACAACTCGACAAGoAA2007 GTGGTTATCGGACTCTACATAATGTCAACG

Example 23: Construction of an Optimized TESA Gene for Expression in C.tropicalis

The gene sequence for the E. coli TESA gene was optimized for expressionin C. tropicalis by codon replacement. A new TESA gene sequence wasconstructed using codons from C. tropicalis with similar usage frequencyfor each of the codons in the native E. coli TESA gene (avoiding the useof the CTG codon due to the alternative yeast nuclear genetic codeutilized by C. tropicalis). The optimized TESA gene was synthesized withflanking BspQI restriction sites and provided in vector plDTSMART-Kan(Integrated DNA Technologies). The vector was designated as pAA287.Plasmid pAA287 was cut with BspQI and the 555 bp DNA fragment was gelpurified. Plasmid pAA073 also was cut with BspQI and the linear DNAfragment was gel purified. The two DNA fragments were ligated togetherto place the optimized TESA gene under the control of the C. tropicalisPOX4 promoter. The resulting plasmid was designated pAA294.

Example 24: Cloning of C. tropicalis DGA1 Gene

DGA1 PCR product was amplified from C. tropicalis 20336 genomic DNAusing primers oAA996 and oAA997, shown in the table below. The PCRproduct was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence. A vector containing the DNA sequence of the DGA1gene was designated pAA299.

Primer Sequence oAA996 ATGACTCAGGACTATAAAGACGATAGTCCTACGTCCACTGAG TTGoAA997 CTATTCTACAATGTTTAATTCAACATCACCGTAGCCAAACCT

Example 25: Cloning of C. tropicalis LRO1 Gene

LRO1 PCR product was amplified from C. tropicalis 20336 genomic DNAusing primers oAA998 and oAA999, shown in the table below. The PCRproduct was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence. A vector containing the DNA sequence of the LRO1gene was designated pAA300.

Primer sequence oAA998 ATGTCGTCTTTAAAGAACAGAAAATC oAA999TTATAAATTTATGGCCTCTACTATTTCT

Example 26: Cloning of C. tropicalis ACS1 Gene and Construction ofDeletion Cassette

ACS1 PCR product was amplified from C. tropicalis 20336 genomic DNAusing primers oAA951 and oAA952, shown in the table below. The PCRproduct was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmthe DNA sequence. One such plasmid was designated pAA275. Plasmid pAA280was digested with BamHI to release a 2.0 kb P_(URA3)URA3T_(URA3)P_(URA3)cassette. Plasmid pAA275 was digested with BglII and gel purified. Thetwo pieces were ligated together to generate plasmid pAA276 and pAA282.Plasmid pAA276 and pAA282 have the P_(URA3)URA3T_(URA3)P_(URA3) cassetteinserted into the ACS gene in opposite orientations.

Primer sequence oAA951 CCTACTTCCACAGCTTTAATCTACTATCAT oAA952TTTAAGAAAACAACTAAGAGAAGCCAC

Example 27: Construction of Strain sAA722 (pox4a::ura3/pox4b::ura3POX5/POX5 ACS1/acs1:: P_(URA3)URA3T_(URA3)P_(URA3))

Plasmid pAA276 was digested with BamHI/XhoI and column purified. StrainsAA329 (ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5) was transformedwith the linearized DNA and plated on SCD-ura plate. Several colonieswere checked for ACS1 disruption. One such strain was designated sAA722.

Example 28: Construction of Strain sAA741 (pox4a::ura3/pox4b::ura3POX5/POX5 ACS1/acs1::P_(URA3))

Strain sAA722 was grown in YPD media overnight and plated on 5-FOAplate. Colonies that grew in the presence of 5-FOA were PCR screened forthe looping out of the URA3 gene leaving behind only the URA3 promoter(P_(URA3)) in the ACS1 site. Out of 30 colonies analyzed, only onestrain showed the correct genetic modification. The strain wasdesignated sAA741.

Example 29: Construction of Strain sAA776 (pox4a::ura3/pox4b::ura3POX5/POX5 acs1::P_(URA3)URA3T_(URA3)P_(URA3)/acs1::P_(URA3))

Plasmid pAA282 was digested with BamHI/XhoI and column purified. StrainsAA741 (see Example 28) was transformed with the linearized DNA andplated on SCD-ura plate. Several colonies were checked for double ACS1knockout by insertional inactivation. One such strain was designatedsAA776.

Example 30: Construction of Strain sAA779 (pox4a::ura3/pox4b::ura3POX5/POX5 acs1:: P_(URA3)/acs1::P_(URA3))

Strain sAA776 (see Example 29) was grown in YPD media overnight andplated on 5-FOA plates. Colonies that grew in the presence of 5-FOA werePCR screened for the looping out of the URA3 gene leaving behind onlythe URA3 promoter (P_(URA3)) in both ACS1 copies. One such strain wasdesignated sAA779.

Example 31: Construction of Strain sAA811 (pox4a::ura3/pox4b::ura3POX5/POX5 acs1::P_(URA3)/acs1::P_(URA3) ura3::3×P_(POX4)P450A19)

Plasmid pAA156 containing a P450A19 integration cassette was digestedwith ClaI and column purified. Strain sAA779 (see Example 30) wastransformed with the linearized DNA and plated on SCD-ura plate. Severalcolonies were checked for P450A19 integration. From those colonies, qPCRwas performed to check the copy number of P450A19 integration. Onestrain, designated sAA811, contained 3 copies of P450A19.

Example 32: Construction of Strain sAA810 (pox4a::ura3/pox4b::ura3POX5/POX5 acs1::P_(URA3)/acs1::P_(URA3) ura3::5×P_(POX4)P450A19ura3::8×P_(POX4)TESA)

Plasmid pAA156 containing a P450-A19 integration cassette was digestedwith ClaI and column purified. Plasmid pAA294 containing a TESAintegration cassette also was digested with ClaI and column purified.Strain sAA779 was cotransformed with both linearized DNAs and plated onSCD-ura plate. Several colonies were checked for both P450A19integration and TESA integration. Colonies that were positive for bothTESA and P450A19 were further analyzed by qPCR. qPCR was performed tocheck the copy number of the P450A19 and TESA integration events. Onestrain, designated sAA810, contained 5 copies of P450A19 and 8 copies ofTESA.

Example 33. General Techniques & Methods (used for Examples 34-55)

Growth Media, Reagents and Conditions

YPD, ScD-ura media and plates, and 5-FOA containing plates were made asdescribed in Methods in Yeast Genetics: a Cold Spring Harbor LaboratoryManual/David C. Amberg, Daniel J. Burke, Jeffrey Strathern, —2005 ed.).

SP92+glycerol was made by adding 6.7 g of Bacto yeast nitrogen basewithout amino acids (BD, Franklin Lakes, N.J., USA), 3.0 g of Bactoyeast extract (BD, Franklin Lakes, N.J., USA), 3.0 g of ammoniumsulfate, 1.0 g of potassium phosphate monobasic, 1.0 g of potassiumphosphate dibasic, and 75 g of glycerol to water to a final volume ofone liter. The media was then filtered sterilized.

TB-low N Media was made by adding 1.7 g Bacto yeast nitrogen basewithout ammonium sulfate, 3 g of Bacto yeast extract, 1 g of potassiumphosphate monobasic and 1 g potassium phosphate dibasic per liter ofwater. The media was filtered sterilized.

Overnight cultures were typically grown in 2 to 5 ml of either ScD-uramedia or YPD media in standard culture tubes at 30 C on a shaker atabout 250 rpm.

Molecular Methods

Gel purifications of DNA fragments were done as recommended by themanufacturer using either the GeneJET Gel Extraction Kit (Fermentas Inc,Glen Burnie, Md., USA) or the Zymoclean Gel DNA Recovery Kit(ZymoResearch, Irvine, Calif., USA).

PCR was performed using either PFU Ultra II DNA Polymerase (AgilentTechnologies,Santa Clara, Calif., USA), Taq DNA polymerase (New EnglandBiolabs, Ipswich, Mass., USA), DreamTaq PCR Master Mix (Fermentas Inc,Glen Burnie, Md., USA) or Quick Load Midas Mix (Monserate, San Diego,Calif., USA). Each enzyme was used according to the manufacturer'sinstructions.

Restriction enzyme digestions were conducted as recommended by eachmanufacturer (New England Biolabs, Ipswich, Mass., USA or Fermentas Inc,Glen Burnie, Md., USA). DNA ligations were conducted using either theRapid Ligation Kit (Fermentas Inc, Glen Burnie, Md., USA) or using T4DNA Ligase (New England Biolabs, Ipswich, Mass., USA) according to themanufacturer's instructions.

Yeast transformations were performed as described in Example 10.

Genomic DNA Preparation

The URA3 gene was obtained from genomic DNA of Candida yeast cultureATCC20336.

Genomic DNA from Candida strain ATCC20336 was prepared as follows: About1.5 ml of an overnight culture of cells was and the pellet wasresuspended in about 200 μl of a solution containing 2% Triton X-100, 1%SDS, 100 mM NaCl, 10 MM Tris pH 8.0, and 1 mM EDTA. About 200 μl of acidwashed glass beads were added with about 200 μl ofphenol:chloroform:isoamyl alcohol (25:24:1) at a pH of about 8.0. Thesample was vortexed for about 2 minutes after which about 200 μl ofwater was added. The sample was then centrifuged at 13000 rpm for about10 minutes. The aqueous layer was transferred to a new microcentrifugetube and an equal volume of chloroform:isoamyl alcohol (24:1) solutionwas added. This sample was vortexed for 10 seconds and then centrifugedat 13000 rpm for about 2 minutes. The aqueous layer was transferred to anew microfuge tube and 1 ml of ethanol was added. The tube was thenplaced at −80° C. for about 15 minutes and then spun at 13000 rpm for 15minutes to pellet the DNA The DNA was washed with 70% ethanol andair-dried. The DNA was then resuspended in about 500 μl of water.

Genomic DNA for Klyveromyces lactis (ATCC8585) was purchased from theAmerican Type Culture Collection (Manassas, Va., USA).

To calculate gene copy number, a qPCR method was used as described byJin et al (Appl. Environ. Microbiol. January 2003 vol. 69, no. 1,495-503). qPCR was performed according to the manufacturer'sinstructions using either the Brilliant III Ultra-Fast SYBR® Green QPCRMaster Mix (Agilent Technologies, Englewood, Colo., USA) or theQuantiTect Multiplex PCR NoROX Kit (Qiagen). Genomic DNA from Candidastrain ATCC20336 or plasmid DNA containing the actin gene from ATCC20336and the gene of interest were used as standards.

Primers and probes used throughout these Examples were made via standardDNA synthesis techniques by Integrated DNA Technologies (Coralville,Iowa, USA).

Example 34: Construction of Cloning Plasmid AA073

The plasmid pAA073 was designed to contain the POX4 promoter andterminator from Candida strain ATCC20336 (this strain is also referredto herein as strain sAA001). This plasmid was derived from the publiclyavailable plasmid pUC19 which contains an ampicillin resistance marker.pAA073 was designed to have two SapI restriction enzyme sites locatedbetween the POX4 promoter and terminator which allows unidirectionalcloning of any gene of interest in tandem with the POX4 promoter. TheCandida strain ATCC20336 URA3 gene including the open reading frame andthe endogenous regulatory regions was also placed into pAA073 as aselection marker for transformants. Plasmid pAA073 allows the directintegration of multiple copies of any gene of interest by digesting theplasmid with a unique restriction enzyme such as SpeI, ClaI or BstZ171.These multiple cloning sites for are contained in the URA3 auxotrophicmarker region and can be selectively be used to avoid cutting the geneof interest (i.e., the DNA sequence for the gene of interest can besearched for particular restriction enzyme cut sites and those enzymescan be avoided). In addition, this plasmid can serve as a template tocreate an antibiotic free-DNA cassette containing the gene of interestand the POX 4 regulatory regions inserted between the 3′ and 5′ regionsof the URA3 gene; this cassette can be PCR amplified using the plasmidas a template, and the isolated PCR product can be inserted into anymicroorganism strain.

A diagram of pAA073 is set forth in FIG. 18 and the sequence of pAA073is set forth as SEQ ID NO: 160.

Example 35: Generic Procedure for Creating Yeast Transformation Plasmidsand Integration Cassettes and Creation of a ZWF1 Gene TransformationPlasmid

One of two procedures was used to generate DNA constructs useful to maketransformed Candida yeast strains that contained either amplified levelsof endogenous genes or exogenous genes inserted into the genomic DNA ofthe Candida yeast host. The following endogenous genes were amplifiedfrom genomic Candida ATCC20336 genomic DNA: fatty alcohol dehydrogenase(“ADH”)-ADH1, 2, 3, 4, 5, 7 and 8; ZWF1 (glucose-6-phosphatedehydrogenase); FAT1 (fatty acyl transporter 1); PEX11 (peroxisomalbiogenesis factor 11); HFD1 and HFD2 (human fatty aldehyde dehydrogenase1 and 2), CPRB (cytochrome p450 reductase B), P450A12-A20 and P450D2(cytochrome p450 oxidases 12-20 and D2); FAT1 (fatty acyl transporter1); and IDP2 (cytoplasmic isocitrate dehydrogenase NADP+). The gene GDP1(glyceraldehyse 3 phosphate dehydrogenase) was obtained fromKlyveromyces lactis genomic DNA and is sometimes referred to as“KIGDP1”. In the case of the ADH1 gene, the alleles were separatelycloned; these alleles are referred to as “ADH1-1 and ADH1-2. Inaddition, the ADH1 allele 1 was cloned as the “short” version and thusis referred to as “ADH1-1short”; the ADH1 allele 2 was cloned as bothshort and regular versions and these genes are referred to as“ADH1-2-short” and “ADH1-2”. For ADH2, two separate genes have beenidentified; each of them was cloned and amplified herein and they arereferred to as “ADH2a” and “ADH2b”. The first procedure (“Procedure 1”)resulted in generating a plasmid that was directly transformed intoyeast; this plasmid contained the antibiotic resistance gene kanamycin.

The second procedure (“Procedure 2”) included all of the steps of thefirst procedure, but added an additional final step to remove theantibiotic resistance gene such that the transformed Candida strain didnot contain any exogenous antibiotic resistance genes.

The first step in Procedure 1 was to amplify the gene of interest fromCandida strain ATCC20336 genomic DNA using appropriately designedprimers and standard PCR techniques as set forth above. The sequence ofeach primer is set forth in TABLE 5, 6 and 7. The amplified gene ofinterest was then inserted into plasmid pCR-Blunt II-Topo (LifeTechnologies, Carlsbad, Calif., USA) using standard techniquesrecommended by the manufacturer. The sequence of the gene or interestwas then verified using standard sequencing techniques. The name of theresulting plasmid for each gene of interest is set forth in TABLE 1under the column labeled “Plasmid 1”. Next, Plasmid 1 was digested withappropriate restriction enzymes to isolate the gene of interest insert.This gene of interest was then inserted into pAA073 (described inExample 35) to create “Plasmid 3” for each gene of interest. The name ofeach Plasmid 3 for each gene of interest is set forth in TABLE 1 in thecolumn labeled “Plasmid 3”. It is possible to clone the PCR fragmentdirectly into Plasmid 3 thereby avoiding construction of Plasmid 1. Eachresulting Plasmid 3 contained the gene of interest under the control ofthe POX 4 promoter and terminator, the URA3 gene and regulatory regions,and the ampicillin resistance marker gene. For some constructs, thisPlasmid 3 was cut in the URA3 gene and the entire linearized plasmid wastransformed into Candida strain ATCC20336. Such transformed Candidastrains contained the ampicillin resistance gene.

In the second procedure, the entire first procedure was followed. Aftercreation of Plasmid 3 however, two PCR reactions were conducted. Thefirst reaction was designed to amplify only the 3′ region of the URA3gene; the amplified fragment was then gel purified. A second PCRreaction amplified, as a single fragment, the POX4 promoter, the gene ofinterest, the POX4 terminator and the 5′ region of the URA3 gene. Thisfragment was also gel purified. The two fragments were fused together byPCR and this PCR product was inserted into plasmid pCR-Blunt II-Topo,this plasmid was transformed into E. coli cells and colonies were thenselected for sequence verification of the plasmid insert. The plasmidcontaining the correct sequence was named and is referred to as “Plasmid4” in Table 1. Plasmid 4 was then used for PCR amplification of theentire URA3′-POX4 promoter-gene of interest-POX4 terminator-URA5′construct and this construct was then used to transform Candida cells.The resulting transformed cells contained the gene of interest but noantibiotic resistance genes were introduced into the strain.

Preparation of a ZWF1 Transformation Plasmid

Procedure 1 described immediately above was used to create this plasmid.The ZWF1 gene was PCR amplified from Candida strain ATCC20336 genomicDNA using primers oAA831 and oAA832. The PCR fragment was gel purified,cloned into the plasmid pCR-Blunt II-Topo (Life Technologies, Carlsbad,Calif., USA) using standard techniques recommended by the manufacturerand the sequences were verified. The plasmid pCR-Blunt II-TOPO containsa kanamycin resistance gene. The resulting plasmid containing the geneencoding the ZWF1 polypeptide was named pAA246 (“Plasmid 1”). The openreading frame of ZWF1 was then cloned as a Sap1 fragment into pAA073.The resulting plasmid was named pAA253 (“Plasmid₃”)_(.)

Example 36: Creation of an Antibiotic-Free Yeast Integration Cassettefor the ADH2a Gene

Procedure 2 described in the previous Example was used to create anintegration cassette to introduce the gene encoding ADH2a into Candidayeast cells. The ADH2a gene was PCR amplified using standard proceduresfrom Candida strain genomic DNA using primers oAA3018 and oAA3019. ThePCR fragment was gel purified, cloned into pCR-Blunt II-Topo(Invitrogen, Carlsbad, Calif., USA) using standard cloning techniquesand the sequence was verified. Plasmid 1 containing the correct sequencewas named pAA671. The ADH2A fragment from pAA671 was then subcloned intopAA073 using SapI restriction enzyme sites to form Plasmid 3, referredto as pAA683, which places the ADH2a open reading frame under thecontrol of the POX4 promoter and POX4 terminator. An antibiotic-freecassette was then created by assembly PCR. The 3′ region of URA3 and aseparate fragment containing the POX4 promoter, ADH2a open readingframe, POX4 terminator, and 5′ region URA3 were each amplified using PCRwith either primers oAA2206 and oAA2207, or with primers oAA2208 andoAA2209, respectively. The PCR products were gel-purified, combined andre-amplified using primers oAA2206 and oAA2209. The resulting PCRfragment was cloned into pCR-Blunt II Topo (Life Technologies, Carlsbad,Calif., USA) and sequence verified. A plasmid with the correct sequence(“Plasmid 4”) was named pAA711.

Example 37: Creation of an Antibiotic-free Yeast Integration Cassettefor the K. lactis GDP1 Gene

KIGDP1 was cloned from genomic DNA at the same time that it wasmutagenized to replace an internal CUG codon to another leucine encodingcodon by replacing guanosine at position 774 with an adenosine. The 5′region or 3′ region of KIGDP1 was PCR amplified from K. lactis genomicDNA using either oAA2457 and oAA2459 or oAA2458 and oJHR4, respectively.The PCR fragments were gel purified and combined to be used as templatefor a PCR amplification with oAA2457 and oJHR4. The PCR fragment was gelpurified and cloned into pCR-Blunt II-TOPO as recommended by themanufacturer. Plasmids were sequenced and a plasmid with the rightsequence named pAA541. This plasmid was the template for the PCR withprimers oAA2854 and oAA2855 to create plasmid pAA578. All otherprocedures for preparing this cassette were as described for the ADH2ausing appropriate primers for cloning and gene amplification.

-   >GDP1, KI—SEQ ID NO: 71

Example 38: Other Gene Amplification Cassette Constructs

In addition to ZWF1 and ADH2a, several other genes were placed intoeither transformation plasmids or amplification cassettes using eitherProcedure 1 (transformation plasmids) or Procedure 2 (amplificationcassettes) above. The genes included in these plasmids or cassettes areset forth in TABLE 1. The genes that were inserted into antibiotic-freeamplification cassettes have a Plasmid 4 on the Table 1; those genesthat were put into transformation plasmids do not have Plasmid 4. Tables5-8 list the oligonucleotides and oligonucleotide sequences that wereused to subclone and clone the genes described in the Examples herein.

Example 39: Creation of a Candida Strain Overexpressing ZWF1

Plasmid pAA253 was digested with the restriction enzyme ClaI. Thelinearized plasmid was transformed into Candida strain sAA103 usingstandard transformation procedures. Transformants were selected bygrowth in ScD-ura plates using standard procedures. Plates were streakedto generate single colonies and transformants were verified by PCR andsequence analysis. ZWF1 copy number was determined using qPCR. A strainwith approximately six copies of ZWF1 was designated as sAA1233.

Example 40: Creation of a Candida Strain Overexpressing ADH2a

A 3′URA3-P_(POX4)-ADH2A-T_(POX4)-5′URA3 fragment was constructed byusing plasmid pAA711 as a template and PCR amplifying the desired regionof the plasmid with of primers oAA2206 and oAA2209. The PCR fragment wasgel-purified and transformed into Candida strain sAA103. Transformantswere selected by growth in ScD-ura plates. Colonies were streaked forsingle isolates and transformant isolates were verified by PCR. Genecopy number was then determined by qPCR. A strain was identified withapproximately seven copies of P_(POX4)-ADH2A-T_(POX4)4 and was namedsAA1803.

Example 41: Creation of Additional Candida Strains

Several other transformation plasmids or amplification cassettes weregenerated and were transformed in to Candida strain sAA103 usingProcedure 1 or Procedure 2 described above to create novel plasmids andCandida strains. The genes, plasmid names and strain names are set forthin TABLE 1.

TABLE 1 Plasmid Plasmid Plasmid Plasmid 4 Gene 1 2 3 Plasmid 2 StrainADH1-1- pAA698 short ADH1-2 pAA670 pAA682 pAA716 sAA1817 ADH1-2- pAA697pAA700 pAA728 sAA1848 short ADH2A pAA671 pAA683 pAA711 sAA1803 ADH2BpAA672 pAA691 pAA717 sAA1805 ADH7 pAA673 pAA692 pAA714 sAA1841 ADH5pAA674 pAA693 pAA718 sAA1844 ADH3 pAA675 pAA715 pAA730 pAA739 sAA1901ADH4 pAA676 pAA694 pAA719 sAA1839 SFA1 pAA680 pAA699 pAA727 sAA1808 ADH8pAA729 pAA738 pAA741 sAA1904 ZWF1 pAA246 pAA253 sAA1233 FAT1 pAA635PEX11 N/A pAA336 HFD1 pAA677 HFD2 pAA678 pAA695 pAA712 sAA1819 CPRB N/ApAA218 pAA391 P450 Al2 pAA139 pAA151 P450 A13 pAA140 pAA152 P450 A14pAA141 pAA153 pAA367 P450 A15 pAA160 P450 A16 pAA161 P450 A17 pAA142pAA154 P450 A18 pAA143 pAA155 P450 A19 pAA144 pAA156 pAA392 P450 A20pAA145 pAA157 P450 D2 pAA146 pAA158 FAT1 S244A pAA637 FAT1 D495A pAA639IDP2 pAA462 sAA1306 KIGDP1 pAA578 pAA581 pAA592 sAA1485 Note: “Plasmid1”, “Plasmid 3”, and “Plasmid 4” are as described in Example 35;“Plasmid 2” was generated only for the gene alcohol dehydrogenase 3 inwhich the guanosine at position 600 was mutated to an adenosine by sitedirected mutagenesis. To prepare this plasmid, 30 to 50 ng of pAA675 wasused as template in a 50 μl PCR reaction using primers oAA3073 andoAA3074 and PFU Ultra II DNA Polymerase (Agilent Technologies, SantaClara, California, USA) as recommended by manufacture. After the PCR wascompleted, 20 units of Dpn1 (New England Biolabs, Ipswich,Massachusetts, USA) was added to the PCR reaction and incubated for 2hours at 37° C. 5 μl of the reaction was used to transform DH5α cells(Monserate Biotechnology, San Diego CA USA) as recommended bymanufacture. The resulting plasmids were sequence verified, and aplasmid with the right sequence was named pAA715.

Example 42: Creation of Two FAT1 Mutant Genes

Two mutants of the FAT1 gene were created in an attempt to reduce theacyl CoA synthetase activity of the enzyme while maintaining its fattyacid transport activity. The first mutant substituted an alanine atposition 244 for the native serine; the second mutant substituted analanine at position 495 for the native aspartic acid.

To prepare a gene containing the S244A mutation of FAT1,oligonucleotides oAA2839 and oAA2805 were used to amplify the 5′ end ofthe native FAT1 gene from Candida ATCC20336 genomic DNA, whileoligonucleotides oAA2804 and oAA2875 were used to amplify the 3′ end ofthe gene. Both products were gel purified and used as templates for asecond round of PCR using oligonucleotides oAA2839 and oAA2875. Theresultant PCR product was digested along with pAA073 using therestriction enzyme BspQI (New England Biolabs) and the gel purifiedproducts were ligated with T4 DNA ligase (Fermentas). The ligations weretransformed into E. coli DH5α (Montserrat) and plated on LB ampicillin.Minipreps (Qiagen) were completed on several colonies and sequenceconfirmed.

The above process was repeated for the FAT1 D495A mutant gene usingoAA2839 and oAA2842 for the 5′ end of the gene and oAA2841 and oAA2875for the 3′ end. The two ends of the gene were used as described withoAA2839 and oAA2875 to make the full product, digested, cloned andverified as above.

Each mutant gene was inserted into plasmid pAA073.

Example 43: Preparation of Candida Strains Containing Multiple AmplifiedGenes

In addition to creating novel Candida strains in which a single gene wasamplified, several strains were created with more than one geneamplified. These strains were generated by co-transforming strain sAA103with the individual transformation plasmids or amplification cassettesfor each of the genes of interest. TABLE 2 below sets forth the name ofeach such Candida strain created and the genes transformed into thestrain.

TABLE 2 Source Source Final Strain Gene 1 of Gene Gene 2 of Gene Gene 3Plasmid sAA1082 CPRB pAA391 P450 A19 pAA392 sAA1569 CPRB pAA391 p450 A14pAA367 sAA1633 CPRB pAA391 p450 A19 pAA392 ZWF1 pAA246 sAA1644 CPRBpAA391 p450 A19 pAA391 IDP2 pAA462 sAA1304 CPRB pAA391 p450 A19 pAA392

Example 44: Creation of a FAT1 Knockout Strain

To create a Candida strain with decreased FAT1 gene expression, knockout cassettes for each FAT 1 allele were generated. For the firstallele, the 5′ homology region (nucleotides 27 to 488 of the openreading frame of FAT1) was amplified using primers oAA2055 and oAA2056with Candida strain ATCC20336 genomic DNA as a template. The 3′ homologyregion (consisting of nucleotides 1483 to 1891 of the FAT1 open readingframe) was amplified using primers oAA2069 and oAA2060 from the samegenomic DNA. A cassette containing the URA3 marker with the promoterrepeated at the 3′ end was amplified from pAA298 to contain overlapswith both homology regions with oAA2057 and oAA2068. These three DNAfragments pieces were then used in a subsequent PCR reaction to generatethe deletion cassette using oligos oAA2055 and oAA2060. The PCR purifiedcassette was then transformed into strain sAA103 and transformantsverified by PCR to obtain sAA919. This strain was plated on 5FOA to curethe URA3 marker ad was verified by PCR. This strain without URA3 wasdesignated as sAA986.

The second FAT1 allele disruption cassette was generated as follows: A5′ homology region (nucleotides 487 to 951 of the open reading frame)was amplified using primers oAA2070 and oAA2071. A 3′ homology region(nucleotides 987 to 1439 of the open reading frame) was amplified usingprimers oAA2074 and oAA2075 and Candida ATCC20336 genomic DNA as atemplate. A cassette containing the URA3 marker with the promoterrepeated at the 3′ end was constructed to have overlaps with homology toprimers oAA2072 and oAA2073. The three fragments were then used in asubsequent PCR reaction to generate the deletion cassette using oligosoAA2070 and oAA2075. This purified product was then used to transformsAA986, and transformants were verified by PCR as having the secondallele disrupted. A strain with the correct genotype was named sAA1000.This strain was plated on 5FOA and was verified for removal of the URA3marker using PCR. This strain was designated as sAA1182.

Example 45: Creation of a FAT1/ACS1 Double Deletion Strain

Functional POX5 alleles were restored in Candida strain sAA003 bytransformation of sAA003 with POX5 linear DNA to replace theURA3-disrupted loci with a functional allele. A 2,584 bp DNA fragmentwas amplified by PCR using primers oAA0179 and oAA0182 that containedthe POX5 ORF as well as 456 bp upstream and 179 bp downstream of the ORFusing plasmid pAA049 as template. The purified PCR product was used totransform competent sAA003 cells which were plated on YNB-agar platessupplemented with dodecane vapor as the carbon source (e.g., by placinga filter paper soaked with dodecane in the lid of the inverted petridish) and incubated at 30° C. for 4-5 days. Colonies growing on dodecaneas the sole carbon source were restreaked onto YPD-agar and incubated at30° C. Single colonies were grown in YPD cultures and used for thepreparation of genomic DNA. PCR analysis of the genomic DNA preparedfrom the transformants was performed with oligos oAA0179 and oAA0182. Anura3-disrupted POX5 would produce a PCR product of 4,784 bp while afunctional POX5 would produce a PCR product of 2,584 bp. In theresulting strain, sAA235, a PCR product of 2,584 bp was amplifiedindicating that both POX5 alleles had been functionally restored. Anunintended consequence of the selection strategy (YNB-agar withdodecane) was that the cells reverted back to an Ura⁺ phenotype. Withoutbeing limited by any theory, it is believed the absence of uracil in thesolid media and the replacement of the only functional URA3 forced thecells to mutate one of the other ura3 loci back to a functional allele.Plasmid pAA276 was digested with BamHI/XhoI and column purified. StrainsAA329 (ura3/ura3 pox4a::ura3/pox4b::ura3 POX5/POX5) was transformedwith the linearized DNA and plated on SCD-ura plate. Several colonieswere checked for ACS1 disruption. One such strain was designated sAA722.

Strain sAA722 was grown in YPD media overnight and plated on 5-FOAplate. Colonies that grew in the presence of 5-FOA were PCR screened forthe looping out of the URA3 gene leaving behind only the URA3 promoter(P_(URA3)) in the ACS1 site. Out of 30 colonies analyzed, only onestrain showed the correct genetic modification. The strain wasdesignated sAA741. Plasmid pAA282 was digested with BamHI/XhoI andcolumn purified. Strain sAA741 was transformed with the linearized DNAand plated on SCD-ura plate. Several colonies were checked for doubleACS1 knockout by insertional inactivation. One such strain wasdesignated sAA776.

Strain sAA776 was grown in YPD media overnight and plated on 5-FOAplate. Colonies that grew in the presence of 5-FOA were PCR screened forthe looping out of the URA3 gene leaving behind only the URA3 promoter(PURA3) in both ACS1 gene alleles. One such strain was named sAA779. Thefull-length coding sequence of the Fat1 gene was amplified from Candidstrain ATCC20336 genomic DNA using primers oAA1023 and oAA1024. The2,086 bp PCR product was gel purified and ligated into pCR-Blunt II-TOPO(Invitrogen), transformed into competent TOP10 E. coli cells(Invitrogen) and clones containing PCR inserts were sequenced to confirmcorrect DNA sequence. One such plasmid was named pAA296.

Deletion of each FAT1 allele was achieved by transforming cells withlinear DNA cassettes constructed by overlap extension PCR (OE-PCR). Thedeletion cassette for the first FAT1 allele in sAA779 was created fromthree DNA fragments. The first DNA fragment (FAT1 5′ homology) wasamplified from plasmid pAA296 using primers oAA2055 and oAA2056. Thesecond DNA fragment (PURA3URA3TURA3PURA3) was amplified from plasmidpAA298 using primers oAA2057 and oAA2068. A diagram of plasmid pAA298 isset forth in FIG. 19 and the sequence of this plasmid is set forth asSEQ ID NO: 161.

-   >PAA298—SEQ ID NO: 161

The third DNA fragment (FAT1 3′ homology) was amplified from plasmidpAA296 using primers oAA2069 and oAA2060. The location of primerannealing sites in pAA296 that amplify FAT1 DNA fragments are shown inFIG. 59. All three DNA fragments were combined in the same reaction togenerate the full-length deletion cassette by OE-PCR using primersoAA2055 and oAA2060. Strain sAA779 was transformed with the full-lengthdeletion cassette and plated on SCD-Ura plate. Several colonies werescreened by PCR for integration of the deletion cassette at the firstFAT1 allele. One such strain was named sAA865.

Strain sAA865 was grown in YPD media overnight and plated on 5-FOAplate. Colonies that grew in the presence of 5-FOA were PCR screened forthe looping out of the URA3 gene leaving behind only the URA3 promoter(PURA3) in the first FAT1 allele. One such strain was named sAA869.

The deletion of the second FAT1 allele in sAA869 was performed bytransformation with a deletion cassette created by OE-PCR. The deletioncassette for the second FAT1 allele was constructed from three DNAfragments. The first DNA fragment (FAT1 5′ homology) was amplified fromplasmid pAA296 using primers oAA2070 and oAA2075. The second DNAfragment (PURA3URA3TURA3PURA3) was amplified from plasmid pAA298 usingprimers oAA2072 and oAA2073. The third DNA fragment (FAT1 3′ homology)was amplified from plasmid pAA296 using primers oAA2074 and oAA2075. Allthree DNA fragments were combined in the same reaction to create afull-length deletion cassette by OE-PCR using primers oAA2070 andoAA2071. Strain sAA869 was transformed with the full-length deletioncassette and plated on SCD-Ura plate. Several colonies were screened byPCR for integration of the deletion cassette at the second FAT1 allele.One such strain was named sAA875. Candida strain sAA875 was grownovernight in YPD media and then streaked on to in5-fluorotic acidcontaining plates. Single colonies were tested for URA3 reversionfrequency, and the isolate with least reversion frequency was namedsAA886.

A disruption cassette for the first allele of the POX5 gene wasconstructed by overlapping PCR. A 5′ POX5 (+34 to +488 of the ORF) or 3′POX5 (+1487 to +1960 of the OrF) fragment was PCR amplified usinggenomic DNA from ATCC20336 as the template and primers oAA2173 andoAA2174 (for the 5′ fragment) or oAA2177 and oAA2178 (for the 3′fragment). A Candida URA3 gene fragment with direct repeat was PCRamplified using oAA2175 and oAA2176 as primers. The three gene fragmentswere then gel purified, combined, ligated and used as template for tomake the full length construct via PCR using oAA2173 and oAA2178 asprimers. This approximately 2.9 Kb fragment was gel purified and used totransform sAA886. Transformants were selected by growth in ScD-uraplates. Colonies were re-streaked to isolate individual transformants.Disruption of the first allele of POX5 was verified by PCR. A strainwith the right genotype was named sAA940.

Strain sAA940 was grown overnight in YPD and then streaked in5-fluorotic acid containing plates. Strains were screened by PCR for thepresent of the POX5 deletion. A strain with the right genotype wasrenamed sAA969.

A disruption cassette for the second allele of the POX5 gene wasconstructed by overlapping PCR. A 5′ POX5 (+489 to +960 of the ORF) or3′ POX5 (+1014 to +1479 of the ORF) fragment was PCR amplified usinggenomic DNA from ATCC20336 and primers oAA2188 and oAA2189 or oAA2192and oAA2193, respectively. A Candida URA3 gene fragment with theterminator as a direct repeat was PCR amplified using oAA2190 andoAA2191 as primers and pAA298 as template. These three DNA fragmentswere gel purified, combined, ligated and used as template for PCR usingoAA2188 and oAA2193 as primers. This approximately 2.9 Kb fragment wasgel purified and used to transform strain sAA969. Transformants wereselected by growth in ScD-ura plates. Colonies were re-streaked toisolate individual transformants. Strains were screened for disruptionof both POX5 alleles by PCR. A strain with the right genotype was namedsAA988.

Example 46: Construction of a POX4, POX5, ACS1 Deletion Strain

A disruption cassette for the first ACS1 gene allele was constructed byoverlapping PCR. A 5′ ACS1 (+101 to +601 of the ORF) or 3′ ACS1 fragment(+1546 to +1960 of the ORF) was PCR amplified using genomic DNA fromATCC20962 and primers oAA2406 and oAA2407 or oAA2408 and oAA2409,respectively. A Candida URA3 gene fragment was PCR amplified usingoAA2410 and oAA2411 as primers and pAA244 as template. The three genefragments were gel purified, combined, ligated and used as template forPCR using oAA2406 and oAA2409 as primers. This PCR fragment was gelpurified and used to transform sAA103. Transformants were selected bygrowth in ScD-ura plates. Colonies were re-streaked to isolateindividual transformants. Disruption of the first allele of ACS1 wasverified by PCR. A strain with the right genotype was named sAA1185.

sAA1185 was grown overnight in YPD and streaked in streaked in5-fluoorotic acid containing plates. Strains were screened by PCR forthe present of the ACS1 deletion. A strain with the right genotype wasrenamed sAA1313.

A nested disruption cassette was constructed by overlapping PCR. A 5′ACS1 (+626 to +1021 of the ORF) or a 3′ ACS1 (+1151 to +1518 of the ORF)fragment was PCR amplified using genomic DNA from ATCC20336 and primersoAA2412 and oAA2413 or oAA2414 and oAA2415, respectively. A Candida URA3fragment was PCR amplified using oAA2416 and oAA2417 as primers foramplification of the URA3 gene. The three fragments were gel purified,combined and used as template for PCR with oAA2412 and oAA2415 asprimers for this PCR reaction. The correct PCR fragment was gel purifiedand used to transform sAA1184. Transformants were selected by growth inScD-ura plates. Colonies were re-streaked to isolate individualtransformants. These transformants were screened for disruption of bothACS1 alleles by PCR. A strain with the correct genotype was namedsAA1371.

Example 47: Construction and Evaluation of Certain CPR750-CYP450 Strains

Plasmids comprising a combination the CPR750 gene and one or more CYP450genes were created ligating either the CPR750 gene containing theendogenous CPR750 promoter (see plasmid pAA067 in Example 16) into eachof pAA151-158, pAA160 or pAA161 as follows (TABLE 3).

Plasmid pAA151 was digested with Sbf1/SpeI restriction enzymes and the2584 bp fragment encoding CPR750 was isolated and ligated into the 6198bp fragment of pAA067 when digested with Sbf1 and Spe1. The ligationmixture was transformed into E. coli cells (DH5alpha). Plasmids wereverified by restriction enzyme analysis and sequencing. A plasmid withthe correct sequence was named pAA223.

TABLE 3 Frament Frament Final P450 size CPR750 size Plasmid P450 PlasmidEnzymes used Plasmid Enzymes used pAA223 A12 pAA151 Sbf1/SpeI 2584pAA067 Sbf1/SpeI 6198 pAA224 A13 pAA152 Sbf1/SpeI 2609 pAA067 Sbf1/SpeI6198 pAA225 A14 pAA153 Sbf1/SpeI 2581 pAA067 Sbf1/SpeI 6198 pAA226 A15pAA160 Sbfl/Pcil 5712 pAA067 Sbfl/Pcil/ApaL1 3121 pAA227 A16 pAA161Sbfl/Pcil 5712 pAA067 Sbfl/Pcil 3121 pAA228 A17 pAA154 Sbf1/SpeI 2594pAA067 Sbf1/SpeI 6198 pAA229 A18 pAA155 Sbf1/SpeI 2566 pAA067 Sbf1/SpeI6198 pAA230 A19 pAA156 Sbf1/SpeI 2551 pAA067 Sbf1/SpeI 6198 pAA231 A20pAA157 Sbf1/SpeI 2551 pAA067 Sbf1/SpeI 6198 pAA232 D2 pAA148 Sbf1/SpeI3512 pAA067 Sbf1/SpeI 6198

Plasmids pAA223 and pAA233 were linearized with SpeI (New EnglandBiolabs) while the remaining plasmids were linearized with ClaI (NewEngland Biolabs). sAA103 was transformed with the linearized plasmids.Transformants were selected by growth in ScD-ura plates. Colonies werestreaked for single isolates and transformants in each isolate wereselected and verified by PCR.

The strains prepared above were then tested for production of di-acidsusing coconut oil as a substrate (“feedstock”).

Strains were grown overnight in SP92+glycerol (5 mL), then transferredto 50 mL SP92+glycerol (50 mL) at a starting OD=0.4. Each strain wascentrifuged and the pellet resuspended in TB IowN medium (12.5 mL). Toeach flask 2% coconut oil was added. Flasks were incubated at 300 RPMs30° C. Samples (1 mL) were taken at 30 and 96 hrs for GC analysis.

As can be seen in TABLE 4, P450 A19 showed the biggest improvement indiacid formation on C10, C12 and C14 fatty acids.

TABLE 4 Gene Diacid formed from total acid at 30 hrs Strain P450 C6 C8C10 C12 C14 sAA003 N/A 0.24 0.03 0.61 0.40 0.16 sAA0797 P450 A12 0.210.04 0.31 0.11 0.04 sAA0798 P450 A13 0.15 0.03 0.71 0.71 0.35 sAA0799P450 A14 0.18 0.03 0.29 0.08 0.03 sAA0800 P450 A15 0.13 0.04 0.60 0.350.14 sAA0801 P450 A15 0.16 0.06 0.75 0.65 0.33 sAA0802 P450 A15 0.200.08 0.75 0.67 0.38 sAA0803 P450 A16 0.20 0.03 0.67 0.46 0.19 sAA0804P450 A17 0.26 0.07 0.74 0.64 0.41 sAA0805 P450 A18 0.19 0.08 0.81 0.810.55 sAA0806 P450 A19 0.24 0.56 0.95 0.92 0.73 sAA0807 P450 A20 0.220.38 0.83 0.64 0.32

Example 48. Conversion of Methyl Laurate and Methyl Myristate to theCorresponding Diacid—Comparison of Strain sAA1304 to sAA003

A preculture of 80 mL SP92 (6.7 g/L yeast nitrogen base, 3.0 g/L yeastextract, 3.0 g/L (NH₄)₂SO₄, 1.0 g/L K₂HPO₄, 1.0 g/L KH₂PO₄, 75 g/Ldextrose) in a 500 mL baffled flask with foam plugs was inoculated with1.0 mL from a frozen glycerol stock of strain sAA003 (beta-oxidationblocked strain) or strain sAA1304 (beta-oxidation blocked strain plusamplified CPRB and CYP52A19) and incubated for 24 h at 30° C. and 250RPM. Fermentation medium (MM1) of composition 27 g/L dextrose, 7.0 g/Lammonium sulfate, 5.1 g/L potassium phosphate monobasic, 1.024 g/Lmagnesium sulfate heptahydrate, 0.155 g/L calcium sulfate dihydrate,0.06 g/L citric acid anhydrous, 0.04 g/L ferrous sulfate heptahydrate,0.0002 mg/L biotin, 1.0 mL trace minerals solution (0.9 g/L boric acid,0.11 g/L cupric sulfate pentahydrate, 0.18 g/L potassium iodide, 0.806g/L manganese sulfate monohydrate, 0.360 g/L sodium molybdate, 0.720 g/Lzinc sulfate), pH 5.8 was filter sterilized and transferred to a sterilefermentation vessel. Growth was initiated with an inoculum of precultureto an initial OD_(600nm)=1.0 and growth conditions of 35° C., 1000 rpm,1 vvm, pH 5.8. Growth continued for approximately 10-12 h at which pointthe conversion phase was initiated by the addition of a bolus of 5 g/Lof feedstock (methyl myristate only), followed immediately by acontinuous feed of feedstock. Because of the toxicity of lauric acid,which is formed during the conversion process by demethylation of methyllaurate at high concentrations, no initial bolus was given. Feedstockfeed rates varied as follows: methyl myristate (Sigma-Aldrich #W272205),1.0 g/L-h for the first 24 h; 1.5 g/L-h from 24 h to termination; methyllaurate (Sigma-Aldrich #W271500), 0.5 g/L-h for the first 24 h; 1.2g/L-h from 24 h to termination. In addition, a co-feed of glucose wasfed at a rate of 1.25 g/L-h when using methyl myristate as substrate orat a rate of 1.0 g/L-h when using methyl laurate as substrate. Atinduction, the temperature was changed to 30° C. and the pH wasmaintained at 6.0 by addition of 6N KOH. The data in FIG. 20 shows theproduction of either dodecanedioic acid from methyl laurate ortetradecanedioic acid from methyl myristate and demonstrates theimproved productivity of strain sAA1306 over sAA003 on both feedstocks.When methyl myristate was used as feedstock, sAA1306 showed anapproximately 25% improvement in productivity over sAA003.

Example 49. Conversion of Methyl Laurate and Methyl Myristate to theCorresponding Diacid—Comparison of Strain sAA1082 to sAA003

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain) or strain sAA1082 (beta-oxidationblocked strain plus amplified CPRB and CYP52A19) and incubated for 24 hat 30° C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was filtersterilized and transferred to a sterile fermentation vessel. Growth wasinitiated with an inoculum of preculture to an initial OD_(600nm)=1.0and growth conditions of 35° C., 1000 rpm, 1 vvm, pH 5.8. Growthcontinued for approximately 10-12 h at which point the conversion phasewas initiated by the addition of a bolus of 5 g/L of feedstock (methylmyristate only), followed immediately by a continuous feed of feedstock.Because of the toxicity of lauric acid, which is formed during theconversion process by demethylation of methyl laurate at highconcentrations, no initial bolus was given. Feedstock feed rates variedas follows: methyl myristate, 1.0 g/L-h for the first 24 h; 1.5 g/L-hfrom 24 h to termination; methyl laurate, 0.75 g/L-h for the first 24 h;1.4 g/L-h from 24 h to termination. In addition, a co-feed of glucosewas fed at a rate of 1.25 g/L-h for all fermentations. At induction, thetemperature was changed to 30° C. and the pH was maintained at 6.0 byaddition of 6N KOH. The data in FIG. 21 show the production of eitherdodecanedioic acid from methyl laurate or tetradecanedioic acid frommethyl myristate and demonstrate improved productivity of strain sAA1082over sAA003 on both feedstocks. When methyl laurate was used asfeedstock, sAA1082 demonstrated about 23% productivity improvement oversAA003. With methyl myristate as feedstock, sAA1082 showed anapproximately 37% improvement over sAA003.

Example 50. Conversion of Oleic to Cis-9-Octadecenedioic Acid—Comparisonof Strains sAA1233, sAA1306 and sAA1485 to sAA003

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain), strain sAA1233 (beta-oxidation blockedstrain plus amplified ZWF1), strain sAA1306 (beta-oxidation blockedstrain plus amplified IDP2), or strain sAA1485 (beta-oxidation blockedstrain plus amplified KIGDP1) and incubated for 24 h at 30° C. and 250RPM. Fermentation medium (MM1) at pH 5.8 was filter sterilized andtransferred to a sterile fermentation vessel. Growth was initiated withan inoculum of preculture to an initial OD_(600nm)=1.0 and growthconditions of 35° C., 1000 rpm, 1 vvm, pH 5.8. Growth continued forapproximately 10-12 h at which point the conversion phase was initiatedby the addition of a bolus of 5 g/L of oleic acid (Sigma-Aldrich#W281506), followed immediately by a continuous feed of feedstock at arate of 2.0 g/L-h throughout the conversion phase. In addition, aco-feed of glucose was fed at a rate of 1.25 g/L-h for allfermentations. At induction, the temperature was changed to 30° C. andthe pH was maintained at 6.0 by addition of 6N KOH. The data in FIG. 22are averages of three identical fermentations and show the productioncis-9-octadecenedioic acid (C18:1 diacid) from oleic acid.

All three amplified genes (ZWF1, IDP2, and KIGDP1) code for enzymes thatproduce NADPH during the biochemical reaction and, because of that,increased expression of those enzymes should result in increasedintracellular levels of NADPH. Omega-hydroxy fatty acids (HFAs) areobserved to be produced as a result of incomplete oxidation of the fattyacid feedstock to the corresponding diacid. One reason for thisincomplete oxidation may be reduced levels of NADPH, which is requiredfor the over-oxidation reaction of HFAs by cytochrome P450 Thus,increasing the intracellular pool of NADPH should result in decreasedlevels of HFA in the fermentation broth. The concentrations of HFAsproduced during the omega oxidation of oleic acid by strains sAA003,sAA1233, sAA1306 and sAA1485 are shown in FIG. 23. The results (averagesof three fermentations) demonstrate that all three test strains(sAA1233, sAA1306 and sAA1485) produced lower levels of HFAs than thebase strain, sAA003. Production of HFAs in a commercial diacidfermentation process is undesirable, since it results in lower molaryields and has to be removed during purification of the diacid. Theseresults indicate that amplification of either ZWF1, IDP2 or KIGDP1should result in an improved diacid fermentation having lower levels ofHFAs.

Example 51. Conversion of Methyl Decanoate to Sebacic Acid—Comparison ofStrain sAA1082 to sAA003

Omega-oxidation of decanoic acid to produce sebacic acid by a Candidastrain can be impractical due to the high degree of toxicity of thispotential feedstock (ref). An alternative is to use methyl decanoate,which has very low toxicity. Methyl esters of fatty acids can beconverted to the corresponding diacid by beta-oxidation-blocked strainsof Candida since Candida produces an esterase that demethylates thefatty acid ester during the omega-oxidation process, allowing conversionof methyl decanoate into the non-toxic diacid, sebacic acid.Unfortunately, having an excess of methyl decanoate in the fermentationbroth prior to induction of the enzymes involved in omega-oxidationprocess results in sufficient demethylation of methyl decanoate toproduce toxic levels of decanoic acid, resulting in rapid cell death anda failed fermentation. The standard fermentation procedure would utilizethe feedstock (methyl decanoate) as inducer. However, an alternativewould be to induce with a non-toxic inducer, such as decane, which hasthe same carbon chain-length as decanoic acid, but which does notproduce decanoic acid during bioconversion to sebacic acid. A set offour fermentations was performed to compare decane to methyl decanoateas inducer as well as to compare the beta-oxidation blocked base strain,sAA003 to strain sAA1082, which has amplified CPRB and CYP52A19 genes.In a previous example, sAA1082 demonstrated increased productivity oversAA003 with both methyl laurate and methyl myristate.

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain) or strain sAA1082 (beta-oxidationblocked strain plus amplified CPRB and CYP52A19) and incubated for 24 hat 30° C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was filtersterilized and transferred to a sterile fermentation vessel. Growth wasinitiated with an inoculum of preculture to an initial OD_(600nm)=1.0and growth conditions of 35° C., 1000 rpm, 1 vvm, pH 5.8. Growthcontinued for approximately 10-12 h at which point the conversion phasewas induced by the addition of either: 1) a bolus of 10 g/L of decane(Sigma-Aldrich #457116) for 6 h after which a continuous feed of methyldecanoate (TCI America #D0023) at 0.25 g/L-h was initiated or 2) noaddition of decane. Induction was performed by initiating a continuousfeed of methyl decanoate at 0.25 g/L-h. Because of the volatility ofdecane, the aeration rate was reduced to 0.3 vvm during the 6-hinduction phase with decane as inducer. In addition, a co-feed ofglucose was fed at a rate of 1.25 g/L-h for all fermentations. Atinduction, the temperature was changed to 30° C. and the pH wasmaintained at 6.0 by addition of 6N KOH. The data in FIG. 24 show theproduction of decanedioic acid (sebacic acid) and compare theproductivity of the two strains under the two different inductionconditions. When induced only with methyl decanoate, neither strainsAA003 nor sAA1082 produced significant quantities of sebacic acid overthe course of the fermentation. However, both strains produced sebacicacid when induced with decane prior to beginning a slow feed of methyldecanoate. Strain sAA1082, however, yielded an over four times highertiter of sebacic acid at 84 h fermentation time than strain sAA003,indicating that sAA1082 is a superior strain for diacid production onmethyl decanoate as well as methyl laurate and methyl myristate asfeedstock. One of the reasons why productivity was better with strainsAA1082 induced with decane is illustrated in FIG. 25, which shows theamount of decanoic acid produced under the different fermentationconditions. The only fermentation that did not produce a detectablequantity of the toxic by-product, decanoic acid, was the fermentationwith strain sAA1082 induced with decane. The other three fermentationsproduced between 1 and 4 g/L decanoic acid. Viable cell count datademonstrates the toxicity of decanoic acid. The only fermentation whereviable cell counts remained high throughout the fermentation was thefermentation with strain sAA1082 induced with decane. The other threefermentations lost between 10³-10⁵ viable cells/mL of culture broth.That significant reduction of biologically-active cells resulted in alarge accumulation of both methyl decanoate and dextrose in allfermentations except the fermentation with sAA1082 induced with decane.That fermentation showed little to no accumulation of either methyldecanoate or dextrose. These data indicate that it would probably havebeen possible to use a higher methyl decanoate feed rate than 0.25g/L-h. These results demonstrate that under the right inductionconditions and with an improved production strain, it is possible toproduce significant quantities of sebacic acid from the methyl ester ofa toxic fatty acid.

Other non-toxic inducers, such as alkanes with chain lengths greaterthan C6, fatty acids with chain-lengths greater than C12, various estersof fatty acids greater than C12, triglycerides containing variouschain-length fatty acids, or other non-toxic chemicals containing a longaliphatic chain greater than C6 could be used as a non-toxic inducer.However, as in this example, using an inducer that would not producesebacic acid during the omega-oxidation process, would likely result inan oxidation product that would need to be purified from the desiredproduct, sebacic acid.

The method described in this example—for employing a non-toxic feedstockto induce diacid production from the methyl ester of a toxic fattyacid—could be used with fermentations utilizing methyl laurate asfeedstock. Lauric acid is not as toxic to Candida as is decanoic acid,but care must be exercised in the induction process to feed methyllaurate at a rate sufficient to allow good induction withoutoverfeeding, which would result in the production and accumulation oftoxic levels of lauric acid due to demethylation by esterases.

Example 52. Conversion of Methyl Laurate to DDDA—Comparison of StrainsAA1569 to sAA003

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain), or strain sAA1569 (beta-oxidationblocked strain plus amplified CPRB and CYP52A14), and incubated for 24 hat 30° C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 was filtersterilized and transferred to a sterile fermentation vessel. Growth wasinitiated with an inoculum of preculture to an initial OD_(600nm)=1.0and growth conditions of 35° C., 1000 rpm, 1 vvm, pH 5.8. Growthcontinued for approximately 10-12 h at which point the conversion phasewas initiated by a continuous feed of methyl laurate at a rate of 0.75g/L-h for the first 24 h; 1.5 g/L-h from 24 h to termination. Inaddition, a co-feed of glucose was fed at a rate of 1.25 g/L-h for allfermentations. At induction, the temperature was changed to 30° C. andthe pH was maintained at 6.0 by addition of 6N KOH. The data in FIG. 26are averages of two identical fermentations and show the production ofDDDA and 12-hydroxy-dodecanoic acid (HFA) from methyl laurate. Althoughstrain sAA1569 did not exhibit increased productivity over sAA003, itdid produce less than half the amount of HFA as sAA003. This result islikely due to CYP52A14 exhibiting a greater rate of over-oxidation ofomega-hydroxy dodecanoic acid than the native P450s.

Example 53. Conversion of Methyl Laurate to DDDA—Comparison of StrainssAA1082 and sAA1633 to sAA003

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain), strain sAA1082 (beta-oxidation blockedstrain plus amplified CPRB and CYP52A19) or sAA1633 (beta-oxidationblocked strain plus amplified CPRB, CYP52A19 and ZWF1), and incubatedfor 24 h at 30° C. and 250 RPM. Fermentation medium (MM1) at pH 5.8 wasfilter sterilized and transferred to a sterile fermentation vessel.Growth was initiated with an inoculum of preculture to an initialOD_(600nm)=1.0 and growth conditions of 35° C., 1000 rpm, 1 vvm, pH 5.8.Growth continued for approximately 10-12 h at which point the conversionphase was initiated by a continuous feed of methyl laurate at a rate of0.75 g/L-h for the first 24 h; 1.5 g/L-h from 24 h to termination. Inaddition, a co-feed of glucose was fed at a rate of 1.25 g/L-h for allfermentations. At induction, the temperature was changed to 30° C. andthe pH was maintained at 6.0 by addition of 6N KOH. The data in FIG. 27are averages of two identical fermentations and show the production ofDDDA from methyl laurate. Strain sAA1082 again demonstrated about 23%increase in productivity over sAA003. Strain sAA1633 exhibited an evengreater productivity increase of about 30% over sAA003. This additionalproductivity increase was probably due to the amplification of ZWF1,leading to increased production of NADPH, which provides electrons forthe omega-oxidation pathway.

Example 54. Conversion of Methyl Laurate to DDDA—Comparison of StrainssAA1901, sAA1904, sAA1803 and sAA1805 to sAA003

A preculture of 80 mL SP92 in a 500 mL baffled flask with foam plugs wasinoculated with 1.0 mL from a frozen glycerol stock of strain sAA003(beta-oxidation blocked strain), sAA1901 (beta-oxidation blocked strainplus amplified ADH3), sAA1904 (beta-oxidation blocked strain plusamplified ADH8), strain sAA1803 (beta-oxidation blocked strain plusamplified ADH2a) or sAA1805 (beta-oxidation blocked strain plusamplified ADH2b), and incubated for 24 h at 30° C. and 250 RPM.Fermentation medium (MM1) at pH 5.8 was filter sterilized andtransferred to a sterile fermentation vessel. Growth was initiated withan inoculum of preculture to an initial OD_(600nm)=1.0 and growthconditions of 35° C., 1000 rpm, 1 vvm, pH 5.8. Growth continued forapproximately 10-12 h at which point the conversion phase was initiatedby a continuous feed of methyl laurate at a rate of 0.75 g/L-h for thefirst 24 h; 1.0 g/L-h from 24 h to termination. In addition, a co-feedof glucose was fed at a rate of 1.25 g/L-h for all fermentations. Atinduction, the temperature was changed to 30° C. and the pH wasmaintained at 6.0 by addition of 6N KOH. The data in FIG. 28 show theproduction of DDDA while FIG. 29 shows the production of HFAs frommethyl laurate. Although there was some variability in the productivityof DDDA between strains, strains sAA1901 and sAA1805 produced about thesame amount of HFAs as strain sAA003. Only strains sAA1904 and sAA1803produced significantly lower HFAs than sAA003.

TABLE 5 Supplemental List I of Oligonucleotides used in Examples OligoDesignation Nucleotide sequence (optional) oAA0179GAATTCACATGGCTAATTTGGCCTCGGTTCCAC AACGCACTCAGCATTAAAAA oAA0182GAGCTCCCCTGCAAACAGGGAAACACTTGTCAT CTGATTT oAA0509CACACAGCTCTTCCATAATGTCGTCTTCTCCAT CGT oAA0510CACACAGCTCTTCCCTCTCTTCTATTCTTAGTA CATTCTAACATC oAA0511CACACAGCTCTTCCATAATGTCGTCTTCTCCAT CGT oAA0512CACACAGCTCTTCCCTCTCTTCTATTCTTAGAA CATTCTAACGTC oAA0515CACACAGCTCTTCCATAatggccacacaagaaa tcatcg oAA0516CACACAGCTCTTCCctctcttctattcttacat cttgacaaagacaccatcg oAA0517CACACACCCGGGatgactgtacacgatattatc gccac oAA0518CACACACCCGGGctaatacatctcaatattggc accg oAA0519CACACAGCTCTTCCATAatgactgcacaggata ttatcgcc oAA0520CACACAGCTCTTCCctctcttctattcctaata catctcaatgttggcaccg oAA0521CACACACCCGGGatgattgaacaactcctagaa tattgg oAA0522CACACACCCGGGctagtcaaacttgacaatagc acc oAA0523CACACAGCTCTTCCATAatgattgaacaaatcc tagaatattgg oAA0524CACACAGCTCTTCCctctcttctattcctagtc aaacttgacaatagcacc oAA0525CACACAGCTCTTCCATAatgctcgatcagatct tacattactg oAA0526CACACAGCTCTTCCctctcttctattcctatga catcttgacgtgtgcaccg oAA0527CACACAGCTCTTCCATAatgctcgaccagatct tccattactg oAA0528CACACAGCTCTTCCctctcttctattcctattg catcttgacgtatgccccg oAA0529CACACAGCTCTTCCATAatggctatatctagtt tgctatcgtg oAA0530CACACAGCTCTTCCctctcttctattctcaagt tctagttcggatgtacaccc oAA0694CACACAGCTCTTCCATAATGGCTTTAGACAAGT TAGA oAA0695CACACAGCTCTTCCctctcttctattcCTACCA AACATCTTCTTG oAA0831CACACAGCTCTTCCATAatgtcttatgattcat tcggtgactacgtc oAA0832CACACAGCTCTTCCctctcttctattcttagat cttacctttgacatcggtgtttg oAA1023GATATTATTCCACCTTCCCTTCATT oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA oAA2053CACACAGCTCTTCCATAATGGGCGAAATTCAGA AAA oAA2054CACACAGCTCTTCCCTCTCTTCTATTCCTAGTA GCCCAAGTTTTT oAA2055TGCCATCCTTGGTAGTCAGTTATT oAA2056 CCGAAACAACCGTAGATACCTTTAATGGCTTGTCCTTGGTGTTGA oAA2057 TCAACACCAAGGACAAGCCATTAAAGGTATCTA CGGTTGTTTCGGoAA2060 TGTCGCCATTCAACCAGTAGAT

TABLE 6 Supplemental List II of Oligonucleotides used in Examples OligoDesignation Nucleotide sequence (optional) oAA2068TCCTCGTCCATCTTCAACAAGTCGGTACCG AGCTCTGCGAATT oAA2069AATTCGCAGAGCTCGGTACCGACTTGTTGA AGATGGACGAGGA oAA2070TTGATCCACTGTCTTAAGATTGTCAA oAA2071 CCGAAACAACCGTAGATACCTTTAACCAGAACGAAGTAGCGGAGAAT oAA2072 ATTCTCCGCTACTTCGTTCTGGTTAAAGGTATCTACGGTTGTTTCGG oAA2073 CGACAGACCTCACCGACGTATGGTACCGAG CTCTGCGAATToAA2074 AATTCGCAGAGCTCGGTACCATACGTCGGT GAGGTCTGTCG oAA2075AGGATTTTGCTGTTGGTGGC oAA2127 CACACAGCTCTTCCATAATGGTCGCCGATT CTTTAGToAA2128 CACACAGCTCTTCCCTCTCTTCTATTCTTA AGTGGCCTTCCACAAGT oAA2173ACCAAGTTCAACCCAAAGGAGT oAA2174 CCGAAACAACCGTAGATACCTTTAATCTTCGTCAAAAGTGGCGGT oAA2175 ACCGCCACTTTTGACGAAGATTAAAGGTAT CTACGGTTGTTTCGGoAA2176 AATGTCGAAACCCTTGTCTTCAGGGTACCG AGCTCTGCGAATT oAA2177AATTCGCAGAGCTCGGTACCCTGAAGACAA GGGTTTCGACATT oAA2178CGGACTTTTCACCTCTTTCTCTG oAA2188 CACTGACGAGTTTGTCATCAACAC oAA2189CCGAAACAACCGTAGATACCTTTAAGGTAT CGGTGTCCTTCTTCTTGA oAA2190TCAAGAAGAAGGACACCGATACCTTAAAGG TATCTACGGTTGTTTCGG oAA2191ACAAGTAAGCGGCAGCCAAGGGTACCGAGC TCTGCGAATT oAA2192AATTCGCAGAGCTCGGTACCCTTGGCTGCC GCTTACTTGT oAA2193 ACCAATGTCTCTGGCCAAGCoAA2206 TTCCGCTTAATGGAGTCCAAA oAA2209 TAAACGTTGGGCAACCTTGG oAA2406CAGACTCAAAGGCAACCACTT oAA2407 tttattggagctccaattgtaatatttcggGATGACATACTTGACGGAGGTG oAA2408 aaacaaccataaagctgcttgacaaAGAACGAAGAAGAAACCAAGGC oAA2409 GCAACAATTCAATACCTTTCAAACC oAA2410CACCTCCGTCAAGTATGTCATCccgaaata ttacaattggagctccaataaa

TABLE 7 Supplemental List III of Oligonucleotides used in Examples OligoDesignation Nucleotide sequence (optional) oAA2411GCCTTGGTTTCTTCTTCGTTCTttgtcaag cagctttatggttgttt oAA2412ACAAGAGACAGGGCGGCAAA oAA2413 tttattggagctccaattgtaatatttcggCGGTCAAAGTCTTGACATTGG oAA2414 aaacaaccataaagctgcttgacaaACAAAAGATCTTCTGGGCTGC oAA2415 TTTCAACCAGATTTCACCCTG oAA2416CCAATGTCAAGACTTTGACCGccgaaatat tacaattggagctccaataaa oAA2417GCAGCCCAGAAGATCTTTTGTttgtcaagc agctttatggttgttt oAA2804gatttacaccgcgggtaccaccggtttgcc oAA2805 GGCAAACCGGTGGTACCCGCGGTGTAAATC0AA2839 CACACAGCTCTTCCATAATGTCAGGATTAG AAATAGCCGCTG oAA2854CACACAGCTCTTCCATAATGCCCGATATGA CAAACGAAT oAA2855CACACAGCTCTTCCCTCTCTTCTATTCAAC ACCAGCTTCGAAGTCCTTT 0AA2875CACACAGCTCTTCCCTCTCTTCTATTCCTA CAATTTGGCTTTACCGGTACAAA oAA3016CACACAGTCTTCCATAATGCATGCATTATT CTCAAAATC oAA3017CACACAGCTCTTCCCTCTCTTCTATTCTCA TTTGGAGGTATCCAAGA 0AA3018CACAGAGCTCTTCCATAATGTCAATTCCAA CTACTCA oAA3019CACACAGCTCTTCCCTCTCTTCTATTCTTA CTTAGAGTTGTCCAAGA oAA3020CACACAGCTCTTCCATAATGTCAATTCCAA CTACCCA 0AA3021CACACAGCTCTTCCCTCTCTTCTATTCCTA CTTGGCAGTGTCAACAA oAA3022CACACAGCTCTTCCATAATGACTGTTGACG CTTCTTC oAA3023CACACAGCTCTTCCCTCTCTTCTATTCCTA ATTGCCAAAAGCTTTGT 0AA3024CACACAGCTCTTCCATAATGTCACTTGTCC TCAAGCG oAA3025CACACAGCTCTTCCCTCTCTTCTATTCTTA TGGGTGGAAGACAACTC oAA3026CACACAGCTCTTCCATAATGTCAACTCAAT CAGGTTA oAA3027CACACAGCTCTTCCCTCTCTTCTATTCCTA CAACTTACTTGGTCTAA oAA3028CACACAGCTCTTCCATAATGTCATTATCAG GAAAGAC oAA3029CACACAGCTCTTCCCTCTCTTCTATTCTTA ACGAGCAGTGAAACCAC oAA3030CACACAGCTCTTCCATAATGAGTAAGTCAT ACAAGTT oAA3031CACACAGCTCTTCCCTCTCTTCTATTCCTA CAAAGAGGCACCAATAAA oAA3032CACACAGCTCTTCCATAATGTCCCCACCAT CTAAATT oAA3033CACACAGCTCTTCCCTCTCTTCTATTCTCT ATTGCTTATTAGTGATG oAA3035CACACAGCTCTTCCCTCTCTTCTATTCTCA CCACATGTTGACAACAG oAA3036CACACAGCTCTTCCATAatgtctgaatcaa ccgttggaaaaccaatcacctgtaaagccg oAA3054CACACAGCTCTTCCATAATGTCTGCTAATA TCCCAAAAACTCAAAAAG oAA3073gttaggcttcaacgctattcaaatattgaa aagctacaattgttacattg oAA3074caatgtaacaattgtagcttttcaatattt gaatagcgttgaagcctaac oAA3120CACACAGCTCTTCCATAATGTCCGTTCCAA CTACTCA oAA3121CACACAGCTCTTCCCTCTCTTCTATTCCTA CTTTGACGTATCAACGA oAA1023GATATTATTCCACCTTCCCTTCATT oAA1024 CCGTTAAACAAAAATCAGTCTGTAAA oBS1GGTTTCATAAGCCTTTTCACGGTCTTC oBS2 GAGTTGACAAAGTTCAAGTTTGCTGTC oJRH4AGTCAGTACTCGAGTTAAACACCAGCTTCG AAGTCC

TABLE 8 Supplemental list of genes and the names of the oligonucleotidesused to clone or subclone them. Gene Primer 1 Primer 2 ADH1-1-shortoAA3054 oAA3017 ADH1-2 oAA3016 oAA3017 ADH1-2-short oAA3054 oAA3017ADH2A oAA3018 oAA3019 ADH2B oAA3020 oAA3021 ADH7 oAA3022 oAA3023 ADH5oAA3024 oAA3025 ADH3 oAA3026 oAA3027 ADH4 oAA3028 oAA3029 SFA1 oAA3036oAA3035 ADH8 oAA3120 oAA3121 ZWF1 oAA831 oAA832 FAT1 oAA2839 oAA2875FAO1ΔPTS1 oAA3068 oAA3069 PEX11 oAA2127 oAA2128 HFD1 oAA3030 oAA3031HFD2 oAA3032 oAA3033 CPRB oAA694 oAA695 P450 A12 oAA515 oAA516 P450 A13oAA517 oAA518 P450 A14 oAA519 oAA520 P450 A15 oAA509 oAA510 P450 A16oAA511 oAA512 P450 A17 oAA521 oAA522 P450 A18 oAA523 oAA524 P450 A19oAA525 oAA526 P450 A20 oAA527 oAA528 P450 D2 oAA529 oAA530 IDP2 oAA2053oAA2054 KIGDP1* oAA2854 oAA2855

Example 55. Replacement of the FAO1 Promoter with a Stronger orConstitutive Promoter

The following Promoter Replacement DNA I molecule is constructed byeither overlapping PCR, DNA synthesis or a combination of both from fivedifferent DNA fragments (Pieces A to E) as illustrated in FIG. 30.

Piece A (e.g., SEQ ID NO: 162)=>about 250 bp piece of the 5′untranslated region of Candida strain ATCC20336 FAO1 gene (from aboutposition −500 to about −250).

Piece B (e.g., SEQ ID NO: 163)=>URA3 marker.

Piece C (e.g., SEQ ID NO: 164)=>about 50 bp of unstranslated region ofCandida strain ATCC20336 FAO1 (from position −300 to −250).

Piece D (e.g., SEQ ID NOS: 165, 166, 167)=>500 bp to 1 kb piece of thepromoter of POX4, PEX11 or TEF1 gene, each obtained from Candida strainATCC20336.

Piece E (SEQ ID NO: 168)=>First 250 bp of the coding sequence of FAO1from Candida strain ATCC20336.

This Promoter Replacement DNA I integrates into at least one of thechromosomes but it may also integrate in both chromosomes depending onthe nucleotide sequence divergence between the two chromosomes. Theregion of −1500 to +500 of the FAO1 gene is sequenced for bothchromosomes. The −1500 to +500 area is PCR amplified with primers oBS1and oBS2 using genomic DNA from ATCC20336. The PCR fragment is clonedinto pCR-Blunt II Topo Multiple clones are sequenced and the sequence ofthe second allele is determined. Pieces A, C and E and are changed tomatch the sequence of the second allele. A second promoter replacementcassette is constructed, sequence verified and named PromoterReplacement DNA II.

A Candida strain such as sAA103 is transformed with Promoter ReplacementDNA cassette I. Transformants are selected by growth in ScD-ura plates.Colonies are streaked for single isolates. Correct insertion of theintegrated piece is verified by PCR. A correct strain is grown in YPDovernight and plated in 5-FOA containing plates to select for theloop-out of the URA3 marker. Ura− strains are streaked for singleisolates and loop out of URA3 is verified by PCR. This strain now hasone FAO1 allele under the control of the POX4, PEX11 or TEF1 promoter.

The ura− strain is then transformed with the Promoter Replace DNA IImolecule. Transformants are selected by growth in ScD-ura plates.Colonies are streaked for single isolates. Correct insertion of theintegrated piece in the second sister chromosome is verified by PCR. Acorrect strain is grown in YPD overnight and plated in 5-FOA containingplates to select for the loop-out of the URA3 marker. Ura− strains arestreaked for single isolates and the loop out of URA3 is verified byPCR. This strain now has both alleles under the control of the POX4,PEX11 or TEF1 promoter.

This strain is then tested in fermentation for improved performance ascompatred with a strain not containing this genetic modification. DNAsequences for each fragment used in the constructs are set forth below:

-   SEQ ID NO: 162—Piece A (5′ untranslated region of FAO1 (from    position −500 to −250).-   SEQ ID NO: 163—Piece B—URA3 marker.-   SEQ ID NO: 164—Piece C.-   SEQ ID NO: 165—Piece D—Promoter POX4.-   SEQ ID NO: 166—Piece D—Promoter PEX11.-   SEQ ID NO: 167—Piece D—Promoter TEF1.-   SEQ ID NO: 168—Piece E—First 250 bp of the coding sequence of FAO1.

Example 56—Increasing NADPH Production by Overexpression of CytosolicMAE1 and PYC2

The open reading frame of ScMAE1 (non-mitochondrial) and ScPYC2 aremutagenized to replace any CTG codon with other leucine-encoding codons.Two plasmids are constructed that replaces the Candida HFD2 of pAA712open reading frame with either the ScMAE1 or the ScPYC2 open readingframe. A 3′URA3-P_(POX4)-ScMAE1*−T_(POX4)−5′URA3 or 3′URA3⁻P_(POX4) ⁻ScPYC2-T_(POX4)-5′URA3 fragment are amplified with PCR using primersoAA2206 and oAA2209 and the corresponding plasmid, as template. The twoPCR fragments are gel-purified, combined, and transformed into sAA103.Transformants are selected by growth in ScD-ura plates. Colonies arestreaked for single colonies and transformants verified by PCR and copynumber determined by qPCR. A strain is identified with approximately5-10 copies of P POX4⁻ ScMAE1*-T_(POX4) and 5-10 copies ofP_(POX4)-ScPYC2-T_(POX4) strain.

A parallel approach is taken by replacing ScMAE1(non-mitochondrial) withthe Candida MAE1 (non-mitochondrial). A strain is identified withapproximately 5-10 copies of P_(POX4)-MAE/*T_(POX4) and 5-10 copies of Ppox4-ScPYC2-T_(POX4).

Example 57: Certain Nucleotide and Amino Acid Sequences for GeneticModification

SEQ ID NO:  Description Sequence SEQ ThioesteraseMVAAAATSAFFPVPAPGTSPKPGKSGNWPSSLSPTFKPKSIPNAGFQVKA ID activityNASAHPKANGSAVNLKSGSLNTQEDTSSSPPPRAFLNQLPDWSMLLTAIT NO:  CupheaTVFVAAEKQWTMLDRKSKRPDMLVDSVGLKSIVRDGLVSRQSFLIRSYEI 1 lanceolataGADRTASIETLMNHLQETSINHCKSLGLLNDGFGRTPGMCKNDLIWVLTK Amino acid (A.A.MQIMVNRYPTWGDTVEINTWFSQSGKIGMASDWLISDCNTGEILIRATSV Seq)WAMMNQKTRRFSRLPYEVRQELTPHFVDSPHVIEDNDQKLHKFDVKTGDSIRKGLTPRWNDLDVNQHVSNVKYIGWILESMPIEVLETQELCSLTVEYRRECGMDSVLESVTAVDPSENGGRSQYKHLLRLEDGTDIVKSRTEWRPKNA GTNGAISTSTAKTSNGNSASSEQ FAO-13 (fattyatggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcID alcohol oxidaseatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaagtacgaaNO:  activity)gagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacacag 2C. TropicalistcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttgNucleotide (Nuc.gctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggSeq)cctcgtggcgcgactccccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag SEQ FAO-13 (fattyMAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVR ID alcohol oxidaseTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPI NO:  activity)KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY 3 C. TropicalisPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVV A.A. SeqAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ FAO-17(fattyatggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcID alcohol oxidaseatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccgacaaatacg NO: activity)aggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacac 4C. TropicaliscgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgttttgggatcaagggtcttNuc. Seqggcaccagctttgaccaactcgttgactcctatcaaggacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggccccaaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattcaagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgcaagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag SEQ FAO-17(fattyMAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRT ID alcohol oxidaseFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIK NO:  activity)DMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYP 5 C. TropicalisGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVA A.A. SeqHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKI SEQ FAO-20(fattyatggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcID alcohol oxidaseatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaagtacgaaNO:  activity)gagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacacag 6C. TropicalistcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttgNuc. Seqgctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaaattgttcaggttggtttccacgcttaccttggttactttcacgagattggccaatgagttgcatttgaaagccattcactatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgaccctttcaagtaccagtttatggaaaagccaaagtttgacggcgctgagttgtacttgccagatattgatgttatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag SEQ FAO-20(fattyMAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEYVR ID alcohol oxidaseTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALTNSLTPI NO:  activity)KDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHY 7 C. TropicalisPGREDREKAYETQEIDPFKYQFMEKPKFDGAELYLPDIDVIIIGSGAGAGV A.A. SeqVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ FAO-2a(fattyatgaataccttcttgccagacgtgctcgaatacaaacacgtcgacacccttttgttattgtgtgacgggatcID alcohol oxidaseatccacgaaaccacagtcgatcagatcaaggacgccattgctcccgacttccctgaggaccagtacgaNO:  activity)ggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagccgtctacgacacgat 8C. TropicaliscaacgccaccccaaccgatgccgtgcacatgtgtattgtcttgaccaccgcattggactccagaatcttgNuc. Seqgcccccacgttgaccaactcgttgacgcctatcaaggatatgaccttgaaggagcgtgaacaattgttggcctcttggcgtgattccccgattgcggcaaagagaagattgttcagattgatttcctcgcttaccttgacgacgtttacgagattggccagcgaattgcacttgaaagccatccactaccctggcagagacttgcgtgaaaaggcgtatgaaacccaggtggttgaccctttcaggtacctgtttatggagaaaccaaagtttgacggcgccgaattgtacttgccagatatcgacgtcatcatcattggatcaggcgccggtgctggtgtcatggcccacactctcgccaacgacgggttcaagaccttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgacgctgatggcgtgaaagagttgtaccaaggtaaaggtgctttggccaccaccaatcagcagatgtttattcttgccggttccactttgggcggtggtaccactgtcaactggtctgcttgccttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtcttgaatttgctgccgatgaagcctacgacaaagcgcaggattatgtttggaaacaaatgggtgcttcaacagatggaatcactcactccttggccaacgaagttgtggttgaaggaggtaagaagttgggctacaagagcaaggaaattgagcagaacaacggtggccaccctgaccacccatgtggtttctgttacttgggctgtaagtacggtattaaacagggttctgtgaataactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctcaacaagaatggcgtcgcttatggtatcttgtgtgaggatgtcgaaaccggagtcaggttcactattagtggccccaaaaagtttgttgtttctgctggttctttgaacacgccaactgtgttgaccaactccggattcaagaacaagcacattggtaagaacttgacgttgcacccagtttccaccgtgtttggtgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctttgttacgaggttgctgacttggacggcaagggccacggatgcagaatcgaaaccatcttgaacgctccattcatccaagcttctttgttgccatggagaggaagtgacgaggtcagaagagacttgttgcgttacaacaacatggtggccatgttgcttatcacgcgtgataccaccagtggttcagtttctgctgacccaaagaagcccgacgctttgattgtcgactatgagattaacaagtttgacaagaatgccatcttgcaagctttcttgatcacttccgacatgttgtacattgaaggtgccaagagaatcctcagtccacagccatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacgatcaaggacaaggactatgttgagtggagagccaaggctgctaagatacctttcgacacctacggttctgcatatgggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggtgctgttgatactgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaacccaatgatatccaccatgacctttgctagacagattgcgttaggtttggctgactccttgaagaccaaacccaagttgtag SEQFAO-2a(fatty MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIKDAIAPDFPEDQYEEYLKT IDalcohol oxidase FTKPSETPGFREAVYDTINATPTDAVHMCIVLTTALDSRILAPTLTNSLTPIKNO:  activity) DMTLKEREQLLASWRDSPIAAKRRLFRLISSLTLTTFTRLASELHLKAIHYP 9C. Tropicalis GRDLREKAYETQVVDPFRYSFMEKPKFDGAELYLPDIDVIIIGSGAGAGVMA.A. Seq AHTLANDGFKTLVLEKGKYFSNSELNFNDADGVKELYQGKGALATTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTDGITHSLANEVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILNKNGVAYGILCEDVETGVRFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYEINKFDKNAILQAFLITSDMLYIEGAKRILSPQPWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMSTCRMSGKGPKYGAVDTDGRLFECSNVYVADASVLPTASGANPMISTMTFARQIALGLADSLKTKPKL SEQ FAO-2b(fattyatgaataccttcttgccagacgtgctcgaatacaaacacgtcgatacccttttgttattatgtgacgggatcaID alcohol oxidasetccacgaaaccacagtcgaccagatcagggacgccattgctcccgacttccctgaagaccagtacga NO: activity)ggagtatctcaagaccttcaccaagccatctgagacccctgggttcagagaagccgtctacgacacgat 10C. TropicaliscaacagcaccccaaccgaggctgtgcacatgtgtattgtattgaccaccgcattggactcgagaatcttgNuc. Seqgcccccacgttgaccaactcgttgacgcctatcaaggatatgaccttgaaagagcgtgaacaattgttggctgcctggcgtgattccccgatcgcggccaagagaagattgttcagattgatttcctcacttaccttgacgacctttacgagattggccagcgacttgcacttgagagccatccactaccctggcagagacttgcgtgaaaaggcatatgaaacccaggtggttgaccctttcaggtacctgtttatggaaaaaccaaagtttgacggcaccgagttgtacttgccagatatcgacgtcatcatcattggatccggtgccggtgctggtgtcatggcccacactttagccaacgacgggtacaagaccttggttttggaaaagggaaagtatttcagcaactccgagttgaactttaatgatgccgatggtatgaaagagttgtaccaaggtaaatgtgcgttgaccaccacgaaccagcagatgtttattcttgccggttccactttgggcggtggtaccactgttaactggtctgcttgtcttaaaacaccatttaaagtgcgtaaggagtggtacgacgagtttggtcttgaatttgctgccgacgaagcctacgacaaagcacaagactatgtttggaaacaaatgggcgcttctaccgaaggaatcactcactctttggcgaacgcggttgtggttgaaggaggtaagaagttgggttacaagagcaaggaaatcgagcagaacaatggtggccatcctgaccacccctgtggtttctgttacttgggctgtaagtacggtattaagcagggttctgtgaataactggtttagagacgcagctgcccacgggtccaagttcatgcaacaagtcagagttgtgcaaatcctccacaataaaggcgtcgcttatggcatcttgtgtgaggatgtcgagaccggagtcaaattcactatcagtggccccaaaaagtttgttgtttctgcaggttctttgaacacgccaacggtgttgaccaactccggattcaagaacaaacacatcggtaagaacttgacgttgcacccagtttcgaccgtgtttggtgactttggcagagacgtgcaagccgaccatttccacaaatctattatgacttcgctctgttacgaagtcgctgacttggacggcaagggccacggatgcagaatcgagaccatcttgaacgctccattcatccaagcttctttgttgccatggagaggaagcgacgaggtcagaagagacttgttgcgttacaacaacatggtggccatgttgcttatcacccgtgacaccaccagtggttcagtttctgctgacccaaagaagcccgacgctttgattgtcgactatgacatcaacaagtttgacaagaatgccatcttgcaagctttcttgatcacctccgacatgttgtacatcgaaggtgccaagagaatcctcagtccacaggcatgggtgccaatctttgagtcgaacaagccaaaggagcaaagaacaatcaaggacaaggactatgtcgaatggagagccaaggctgccaagatacctttcgacacctacggttctgcctatgggtccgcacatcaaatgtccacctgtcgtatgtccggaaagggtcctaaatacggcgccgttgataccgatggtagattgtttgaatgttcgaatgtctatgttgctgatgctagtgttttgcctactgccagcggtgccaacccaatgatctccaccatgacgtttgctagacagattgcgttaggtttggctgactctttgaagaccaaacccaagttgtag SEQFAO-2b(fatty MNTFLPDVLEYKHVDTLLLLCDGIIHETTVDQIRDAIAPDFPEDQYEEYLKT IDalcohol oxidase FTKPSETPGFREAVYDTINSTPTEAVHMCIVLTTALDSRILAPTLTNSLTPIKNO:  activity) DMTLKEREQLLAAWRDSPIAAKRRLFRLISSLTLTTFTRLASDLHLRAIHYP 11C. Tropicalis GRDLREKAYETQVVDPFRYSFMEKPKFDGTELYLPDIDVIIIGSGAGAGVMA.A. Seq AHTLANDGYKTLVLEKGKYFSNSELNFNDADGMKELYQGKCALTTTNQQMFILAGSTLGGGTTVNWSACLKTPFKVRKEWYDEFGLEFAADEAYDKAQDYVWKQMGASTEGITHSLANAVVVEGGKKLGYKSKEIEQNNGGHPDHPCGFCYLGCKYGIKQGSVNNWFRDAAAHGSKFMQQVRVVQILHNKGVAYGILCEDVETGVKFTISGPKKFVVSAGSLNTPTVLTNSGFKNKHIGKNLTLHPVSTVFGDFGRDVQADHFHKSIMTSLCYEVADLDGKGHGCRIETILNAPFIQASLLPWRGSDEVRRDLLRYNNMVAMLLITRDTTSGSVSADPKKPDALIVDYDINKFDKNAILQAFLITSDMLYIEGAKRILSPQAWVPIFESNKPKEQRTIKDKDYVEWRAKAAKIPFDTYGSAYGSAHQMST SEQ FAO-18(fattyatggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacgggatcID alcohol oxidaseatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccgacaaatacg NO: activity)aggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgtctacaacac 12C. TropicaliscgtcaacgcaaacaccatggatgcaatccaccagttcattatcttgaccaatgttttgggatcaagggtcttNuc. Seqggcaccagctttgaccaactcgttgactcctatcaaggacatgagcttggaagaccgtgaaaagttgttagcctcgtggcgtgactcccctattgctgctaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggggccggtgctggtgtcgtggcccacactttgaccaacgacggcttcaagagtttggttttggaaaagggcagatactttagcaactccgagttgaactttgatgacaaggacggggttcaagaattataccaaagtggaggtactttgaccaccgtcaaccagcagttgtttgttcttgctggttccacttttggtggtggtaccactgtcaattggtcggcctgtcttaaaacgccattcaaggtgcgtaaggaatggtatgatgagtttggcgttgactttgctgccgatgaagcctacgacaaagcacaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggcaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcatcacagatgcggtttctgttatttgggttgtaagcacggtatcaagcagggctctgttaataactggtttagagacgcagctgcccacggttctcagttcatgcaacaggttagagttttgcaaatccttaacaagaagggcatcgcttatggtatcttgtgtgaggatgttgtaaccggtgccaagttcaccattactggccccaaaaagtttgttgttgccgccggcgccttaaacactccatctgtgttggtcaactccggattcaagaacaagaacatcggtaagaacttaactttgcatccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagatcacttccacaactccatcatgactgctctttgttcagaagccgctgatttagacggcaagggtcatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggccatgttacttcttagtcgtgataccaccagtggttccgtttcgtcccatccaactaaacctgaagcattagttgtcgagtacgacgtgaacaagtttgacagaaactccatcttgcaggcattgttggtcactgctgacttgttgtacattcaaggtgccaagagaatccttagtccccaaccatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattccttttgacacctacggctcgccttatggttcggcgcatcaaatgtcttcttgtcgtatgtcaggtaagggtcctaaatacggtgctgttgataccgatggtagattgtttgaatgttcgaatgtttatgttgctgacgctagtcttttgccaactgctagcggtgctaatcctatggtcaccaccatgactcttgcaagacatgttgcgttaggtttggcagactccttgaagaccaaggccaagttgtag SEQ FAO-1(fattyMAPFLPDQVDYKHVDTLMLLCDGIIHETTVDEIKDVIAPDFPADKYEEYVRT ID alcohol oxidaseFTKPSETPGFRETVYNTVNANTMDAIHQFIILTNVLGSRVLAPALTNSLTPIK NO:  activity)DMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLANELHLKAIHYP 13 C. TropicalisGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVA A.A. SeqHTLTNDGFKSLVLEKGRYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPHHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGIAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSSHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQPWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ cytochrome P450atggccacacaagaaatcatcgattctgtacttccgtacttgaccaaatggtacactgtgattactgcagcID A12 (CYP52A12)agtattagtcttccttatctccacaaacatcaagaactacgtcaaggcaaagaaattgaaatgtgtcgatcNO:  Nuc. Seqcaccatacttgaaggatgccggtctcactggtattctgtctttgatcgccgccatcaaggccaagaacgac14ggtagattggctaactttgccgatgaagttttcgacgagtacccaaaccacaccttctacttgtctgttgccggtgctttgaagattgtcatgactgttgacccagaaaacatcaaggctgtcttggccacccaattcactgacttctccttgggtaccagacacgcccactttgctcctttgttgggtgacggtatcttcaccttggacggagaaggttggaagcactccagagctatgttgagaccacagtttgctagagaccagattggacacgttaaagccttggaaccacacatccaaatcatggctaagcagatcaagttgaaccagggaaagactttcgatatccaagaattgttctttagatttaccgtcgacaccgctactgagttcttgtttggtgaatccgttcactccttgtacgatgaaaaattgggcatcccaactccaaacgaaatcccaggaagagaaaactttgccgctgctttcaacgtttcccaacactacttggccaccagaagttactcccagactttttactttttgaccaaccctaaggaattcagagactgtaacgccaaggtccaccacttggccaagtactttgtcaacaaggccttgaactttactcctgaagaactcgaagagaaatccaagtccggttacgttttcttgtacgaattggttaagcaaaccagagatccaaaggtcttgcaagatcaattgttgaacattatggttgccggaagagacaccactgccggtttgttgtcctttgctttgtttgaattggctagacacccagagatgtggtccaagttgagagaagaaatcgaagttaactttggtgttggtgaagactcccgcgttgaagaaattaccttcgaagccttgaagagatgtgaatacttgaaggctatccttaacgaaaccttgcgtatgtacccatctgttcctgtcaactttagaaccgccaccagagacaccactttgccaagaggtggtggtgctaacggtaccgacccaatctacattcctaaaggctccactgttgcttacgttgtctacaagacccaccgtttggaagaatactacggtaaggacgctaacgacttcagaccagaaagatggtttgaaccatctactaagaagttgggctgggcttatgttccattcaacggtggtccaagagtctgcttgggtcaacaattcgccttgactgaagcttcttatgtgatcactagattggcccagatgtttgaaactgtctcatctgatccaggtctcgaataccctccaccaaagtgtattcacttgaccatgagtcacaacgatggtgtctttgtcaagatgtaaSEQ cytochrome P450atgactgtacacgatattatcgccacatacttcaccaaatggtacgtgatagtaccactcgctttgattgcttID A13 (CYP52A13)atagagtcctcgactacttctatggcagatacttgatgtacaagcttggtgctaaaccatttttccagaaacaNO:  Nuc. Seqgacagacggctgtttcggattcaaagctccgcttgaattgttgaagaagaagagcgacggtaccctcata15gacttcacactccagcgtatccacgatctcgatcgtcccgatatcccaactttcacattcccggtcttttccatcaaccttgtcaatacccttgagccggagaacatcaaggccatcttggccactcagttcaacgatttctccttgggtaccagacactcgcactttgctcctttgttgggtgatggtatctttacgttggatggcgccggctggaagcacagcagatctatgttgagaccacagtttgccagagaacagatttcccacgtcaagttgttggagccacacgttcaggtgttcttcaaacacgtcagaaaggcacagggcaagacttttgacatccaggaattgtttttcagattgaccgtcgactccgccaccgagtttttgtttggtgaatccgttgagtccttgagagatgaatctatcggcatgtccatcaatgcgcttgactttgacggcaaggctggctttgctgatgcttttaactattcgcagaattatttggcttcgagagcggttatgcaacaattgtactgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtgcacaagtttgctgactactacgtcaacaaggctttggacttgacgcctgaacaattggaaaagcaggatggttatgtgtttttgtacgaattggtcaagcaaaccagagacaagcaagtgttgagagaccaattgttgaacatcatggttgctggtagagacaccaccgccggtttgttgtcgtttgttttctttgaattggccagaaacccagaagttaccaacaagttgagagaagaaattgaggacaagtttggactcggtgagaatgctagtgttgaagacatttcctttgagtcgttgaagtcctgtgaatacttgaaggctgttctcaacgaaaccttgagattgtacccatccgtgccacagaatttcagagttgccaccaagaacactaccctcccaagaggtggtggtaaggacgggttgtctcctgttttggtgagaaagggtcagaccgttatttacggtgtctacgcagcccacagaaacccagctgtttacggtaaggacgctcttgagtttagaccagagagatggtttgagccagagacaaagaagcttggctgggccttcctcccattcaacggtggtccaagaatctgtttgggacagcagtttgccttgacagaagcttcgtatgtcactgtcaggttgctccaggagtttgcacacttgtctatggacccagacaccgaatatccacctaagaaaatgtcgcatttgaccatgtcgcttttcgacggtgccaatattgagatgtattag SEQcytochrome P450atgactgcacaggatattatcgccacatacatcaccaaatggtacgtgatagtaccactcgctttgattgctID A14 (CYP52A14)tatagggtcctcgactacttttacggcagatacttgatgtacaagcttggtgctaaaccgtttttccagaaacNO:  Nuc. Seqaaacagacggttatttcggattcaaagctccacttgaattgttaaaaaagaagagtgacggtaccctcat16agacttcactctcgagcgtatccaagcgctcaatcgtccagatatcccaacttttacattcccaatcttttccatcaaccttatcagcacccttgagccggagaacatcaaggctatcttggccacccagttcaacgatttctccttgggcaccagacactcgcactttgctcctttgttgggcgatggtatctttaccttggacggtgccggctggaagcacagcagatctatgttgagaccacagtttgccagagaacagatttcccacgtcaagttgttggagccacacatgcaggtgttcttcaagcacgtcagaaaggcacagggcaagacttttgacatccaagaattgtttttcagattgaccgtcgactccgccactgagtttttgtttggtgaatccgttgagtccttgagagatgaatctattgggatgtccatcaatgcacttgactttgacggcaaggctggctttgctgatgcttttaactactcgcagaactatttggcttcgagagcggttatgcaacaattgtactgggtgttgaacgggaaaaagtttaaggagtgcaacgctaaagtgcacaagtttgctgactattacgtcagcaaggctttggacttgacacctgaacaattggaaaagcaggatggttatgtgttcttgtacgagttggtcaagcaaaccagagacaggcaagtgttgagagaccagttgttgaacatcatggttgccggtagagacaccaccgccggtttgttgtcgtttgttttctttgaattggccagaaacccagaggtgaccaacaagttgagagaagaaatcgaggacaagtttggtcttggtgagaatgctcgtgttgaagacatttcctttgagtcgttgaagtcatgtgaatacttgaaggctgttctcaacgaaactttgagattgtacccatccgtgccacagaatttcagagttgccaccaaaaacactacccttccaaggggaggtggtaaggacgggttatctcctgttttggtcagaaagggtcaaaccgttatgtacggtgtctacgctgcccacagaaacccagctgtctacggtaaggacgcccttgagtttagaccagagaggtggtttgagccagagacaaagaagcttggctgggccttccttccattcaacggtggtccaagaatttgcttgggacagcagtttgccttgacagaagcttcgtatgtcactgtcagattgctccaagagtttggacacttgtctatggaccccaacaccgaatatccacctaggaaaatgtcgcatttgaccatgtcccttttcgacggtgccaacattgagatgtattagSEQ cytochrome P450atgtcgtcttctccatcgtttgcccaagaggttctcgctaccactagtccttacatcgagtactttcttgacaacID A15 (CYP52A15)tacaccagatggtactacttcatacctttggtgcttctttcgttgaactttataagtttgctccacacaaggtactNO:  Nuc. Seqtggaacgcaggttccacgccaagccactcggtaactttgtcagggaccctacgtttggtatcgctactccg17ttgcttttgatctacttgaagtcgaaaggtacggtcatgaagtttgcttggggcctctggaacaacaagtacatcgtcagagacccaaagtacaagacaactgggctcaggattgttggcctcccattgattgaaaccatggacccagagaacatcaaggctgttttggctactcagttcaatgatttctctttgggaaccagacacgatttcttgtactccttgttgggtgacggtattttcaccttggacggtgctggctggaaacatagtagaactatgttgagaccacagtttgctagagaacaggtttctcacgtcaagttgttggagccacacgttcaggtgttcttcaagcacgttagaaagcaccgcggtcaaacgttcgacatccaagaattgttcttcaggttgaccgtcgactccgccaccgagttcttgtttggtgagtctgctgaatccttgagggacgaatctattggattgaccccaaccaccaaggatttcgatggcagaagagatttcgctgacgctttcaactattcgcagacttaccaggcctacagatttttgttgcaacaaatgtactggatcttgaatggctcggaattcagaaagtcgattgctgtcgtgcacaagtttgctgaccactatgtgcaaaaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgttcttgtacgagttggctaagcaaaccagagacccaaaggtcttgagagaccagttattgaacattttggttgccggtagagacacgaccgccggtttgttgtcatttgttttctacgagttgtcaagaaaccctgaggtgtttgctaagttgagagaggaggtggaaaacagatttggactcggtgaagaagctcgtgttgaagagatctcgtttgagtccttgaagtcttgtgagtacttgaaggctgtcatcaatgaaaccttgagattgtacccatcggttccacacaactttagagttgctaccagaaacactaccctcccaagaggtggtggtgaagatggatactcgccaattgtcgtcaagaagggtcaagttgtcatgtacactgttattgctacccacagagacccaagtatctacggtgccgacgctgacgtcttcagaccagaaagatggtttgaaccagaaactagaaagttgggctgggcatacgttccattcaatggtggtccaagaatctgtttgggtcaacagtttgccttgaccgaagcttcatacgtcactgtcagattgctccaggagtttgcacacttgtctatggacccagacaccgaatatccaccaaaattgcagaacaccttgaccttgtcgctctttgatggtgctgatgttagaatgtactaa SEQ cytochrome P450atgtcgtcttctccatcgtttgctcaggaggttctcgctaccactagtccttacatcgagtactttcttgacaactID A16acaccagatggtactacttcatccctttggtgcttctttcgttgaacttcatcagcttgctccacacaaagtactNO:  (CYP52A16)tggaacgcaggttccacgccaagccgctcggtaacgtcgtgttggatcctacgtttggtatcgctactccgt18 Nuc. Seqtgatcttgatctacttaaagtcgaaaggtacagtcatgaagtttgcctggagcttctggaacaacaagtacattgtcaaagacccaaagtacaagaccactggccttagaattgtcggcctcccattgattgaaaccatagacccagagaacatcaaagctgtgttggctactcagttcaacgatttctccttgggaactagacacgatttcttgtactccttgttgggcgatggtatttttaccttggacggtgctggctggaaacacagtagaactatgttgagaccacagtttgctagagaacaggtttcccacgtcaagttgttggaaccacacgttcaggtgttcttcaagcacgttagaaaacaccgcggtcagacttttgacatccaagaattgttcttcagattgaccgtcgactccgccaccgagttcttgtttggtgagtctgctgaatccttgagagacgactctgttggtttgaccccaaccaccaaggatttcgaaggcagaggagatttcgctgacgctttcaactactcgcagacttaccaggcctacagatttttgttgcaacaaatgtactggattttgaatggcgcggaattcagaaagtcgattgccatcgtgcacaagtttgctgaccactatgtgcaaaaggctttggagttgaccgacgatgacttgcagaaacaagacggctatgtgttcttgtacgagttggctaagcaaactagagacccaaaggtcttgagagaccagttgttgaacattttggttgccggtagagacacgaccgccggtttgttgtcgtttgtgttctacgagttgtcgagaaaccctgaagtgtttgccaagttgagagaggaggtggaaaacagatttggactcggcgaagaggctcgtgttgaagagatctcttttgagtccttgaagtcctgtgagtacttgaaggctgtcatcaatgaagccttgagattgtacccatctgttccacacaacttcagagttgccaccagaaacactacccttccaagaggcggtggtaaagacggatgctcgccaattgttgtcaagaagggtcaagttgtcatgtacactgtcattggtacccacagagacccaagtatctacggtgccgacgccgacgtcttcagaccagaaagatggttcgagccagaaactagaaagttgggctgggcatatgttccattcaatggtggtccaagaatctgtttgggtcagcagtttgccttgactgaagcttcatacgtcactgtcagattgctccaagagtttggaaacttgtccctggatccaaacgctgagtacccaccaaaattgcagaacaccttgaccttgtcactctttgatggtgctgacgttagaatgttctaa SEQ cytochrome P450atgattgaacaactcctagaatattggtatgtcgttgtgccagtgttgtacatcatcaaacaactccttgcataID A17 (CYP52A17)cacaaagactcgcgtcttgatgaaaaagttgggtgctgctccagtcacaaacaagttgtacgacaacgcNO:  Nuc. Seqtttcggtatcgtcaatggatggaaggctctccagttcaagaaagagggcagggctcaagagtacaacg 19attacaagtttgaccactccaagaacccaagcgtgggcacctacgtcagtattcttttcggcaccaggatcgtcgtgaccaaagatccagagaatatcaaagctattttggcaacccagtttggtgatttttctttgggcaagaggcacactctttttaagcctttgttaggtgatgggatcttcacattggacggcgaaggctggaagcacagcagagccatgttgagaccacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcatattcttaagcacaagggtgaatactttgatatccaggaattgttctttagatttaccgttgattcggccacggagttcttatttggtgagtccgtgcactccttaaaggacgaatctattggtatcaaccaagacgatatagattttgctggtagaaaggactttgctgagtcgttcaacaaagcccaggaatacttggctattagaaccttggtgcagacgttctactggttggtcaacaacaaggagtttagagactgtaccaagctggtgcacaagttcaccaactactatgttcagaaagctttggatgctagcccagaagagcttgaaaagcaaagtgggtatgtgttcttgtacgagcttgtcaagcagacaagagaccccaatgtgttgcgtgaccagtctttgaacatcttgttggccggaagagacaccactgctgggttgttgtcgtttgctgtctttgagttggccagacacccagagatctgggccaagttgagagaggaaattgaacaacagtttggtcttggagaagactctcgtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaatgaaaccttgcgtatttacccaagtgtcccaagaaacttcagaatcgccaccaagaacacgacattgccaaggggcggtggttcagacggtacctcgccaatcttgatccaaaagggagaagctgtgtcgtatggtatcaactctactcatttggaccctgtctattacggccctgatgctgctgagttcagaccagagagatggtttgagccatcaaccaaaaagctcggctgggcttacttgccattcaacggtggtccaagaatctgtttgggtcagcagtttgccttgacggaagctggctatgtgttggttagattggtgcaagagttctcccacgttaggctggacccagacgaggtgtacccgccaaagaggttgaccaacttgaccatgtgtttgcaggatggtgctattgtcaagtttgactag SEQcytochrome P450atgattgaacaaatcctagaatattggtatattgttgtgcctgtgttgtacatcatcaaacaactcattgcctaID A18 (CYP52A18)cagcaagactcgcgtcttgatgaaacagttgggtgctgctccaatcacaaaccagttgtacgacaacgttNO:  Nuc. Seqttcggtatcgtcaacggatggaaggctctccagttcaagaaagagggcagagctcaagagtacaacg 20atcacaagtttgacagctccaagaacccaagcgtcggcacctatgtcagtattctttttggcaccaagattgtcgtgaccaaggatccagagaatatcaaagctattttggcaacccagtttggcgatttttctttgggcaagagacacgctctttttaaacctttgttaggtgatgggatcttcaccttggacggcgaaggctggaagcatagcagatccatgttaagaccacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcatatccttaaacacaagggtgagtactttgatatccaggaattgttctttagatttactgtcgactcggccacggagttcttatttggtgagtccgtgcactccttaaaggacgaaactatcggtatcaaccaagacgatatagattttgctggtagaaaggactttgctgagtcgttcaacaaagcccaggagtatttgtctattagaattttggtgcagaccttctactggttgatcaacaacaaggagtttagagactgtaccaagctggtgcacaagtttaccaactactatgttcagaaagctttggatgctaccccagaggaacttgaaaagcaaggcgggtatgtgttcttgtatgagcttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttgaacatcttgttggcaggaagagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaacccacacatctgggccaagttgagagaggaaattgaacagcagtttggtcttggagaagactctcgtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaagcgttccttaacgaaaccttgcgtgtttacccaagtgtcccaagaaacttcagaatcgccaccaagaatacaacattgccaaggggtggtggtccagacggtacccagccaatcttgatccaaaagggagaaggtgtgtcgtatggtatcaactctacccacttagatcctgtctattatggccctgatgctgctgagttcagaccagagagatggtttgagccatcaaccagaaagctcggctgggcttacttgccattcaacggtgggccacgaatctgtttgggtcagcagtttgccttgaccgaagctggttacgttttggtcagattggtgcaagagttctcccacattaggctggacccagatgaagtgtatccaccaaagaggttgaccaacttgaccatgtgtttgcaggatggtgctattgtcaagtttgactag SEQcytochrome P450atgctcgatcagatcttacattactggtacattgtcttgccattgttggccattatcaaccagatcgtggctcatID A19 (CYP52A19)gtcaggaccaattatttgatgaagaaattgggtgctaagccattcacacacgtccaacgtgacgggtggtNO:  Nuc. Seqtgggcttcaaattcggccgtgaattcctcaaagcaaaaagtgctgggagactggttgatttaatcatctccc21gtttccacgataatgaggacactttctccagctatgcttttggcaaccatgtggtgttcaccagggaccccgagaatatcaaggcgcttttggcaacccagtttggtgatttttcattgggcagcagggtcaagttcttcaaaccattattggggtacggtatcttcacattggacgccgaaggctggaagcacagcagagccatgttgagaccacagtttgccagagaacaagttgctcatgtgacgtcgttggaaccacacttccagttgttgaagaagcatatccttaaacacaagggtgagtactttgatatccaggaattgttctttagatttactgtcgactcggccacggagttcttatttggtgagtccgtgcactccttaaaggacgaggaaattggctacgacacgaaagacatgtctgaagaaagacgcagatttgccgacgcgttcaacaagtcgcaagtctacgtggccaccagagttgctttacagaacttgtactggttggtcaacaacaaagagttcaaggagtgcaatgacattgtccacaagtttaccaactactatgttcagaaagccttggatgctaccccagaggaacttgaaaagcaaggcgggtatgtgttcttgtatgagcttgtcaagcagacgagagaccccaaggtgttgcgtgaccagtctttgaacatcttgttggcaggaagagacaccactgctgggttgttgtcctttgctgtgtttgagttggccagaaacccacacatctgggccaagttgagagaggaaattgaacagcagtttggtcttggagaagactctcgtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaggccgtgttgaacgaaactttgagattacacccaagtgtcccaagaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggccccaacggcaaggatcctatcttgatcaggaaggatgaggtggtgcagtactccatctcggcaactcagacaaatcctgcttattatggcgccgatgctgctgattttagaccggaaagatggtttgaaccatcaactagaaacttgggatgggctttcttgccattcaacggtggtccaagaatctgtttgggacaacagtttgctttgactgaagccggttacgttttggttagacttgttcaggagtttccaaacttgtcacaagaccccgaaaccaagtacccaccacctagattggcacacttgacgatgtgcttgtttgacggtgcacacgtcaagatgtcatag SEQ cytochrome P450atgctcgaccagatcttccattactggtacattgtcttgccattgttggtcattatcaagcagatcgtggctcatID A20 (CYP52A20)gccaggaccaattatttgatgaagaagttgggcgctaagccattcacacatgtccaactagacgggtggtNO:  Nuc. Seqttggcttcaaatttggccgtgaattcctcaaagctaaaagtgctgggaggcaggttgatttaatcatctcccg22tttccacgataatgaggacactttctccagctatgcttttggcaaccatgtggtgttcaccagggaccccgagaatatcaaggcgcttttggcaacccagtttggtgatttttcattgggaagcagggtcaaattcttcaaaccattgttggggtacggtatcttcaccttggacggcgaaggctggaagcacagcagagccatgttgagaccacagtttgccagagagcaagttgctcatgtgacgtcgttggaaccacatttccagttgttgaagaagcatattcttaagcacaagggtgaatactttgatatccaggaattgttctttagatttaccgttgattcagcgacggagttcttatttggtgagtccgtgcactccttaagggacgaggaaattggctacgatacgaaggacatggctgaagaaagacgcaaatttgccgacgcgttcaacaagtcgcaagtctatttgtccaccagagttgctttacagacattgtactggttggtcaacaacaaagagttcaaggagtgcaacgacattgtccacaagttcaccaactactatgttcagaaagccttggatgctaccccagaggaacttgaaaaacaaggcgggtatgtgttcttgtacgagcttgccaagcagacgaaagaccccaatgtgttgcgtgaccagtctttgaacatcttgttggctggaagggacaccactgctgggttgttgtcctttgctgtgtttgagttggccaggaacccacacatctgggccaagttgagagaggaaattgaatcacactttgggctgggtgaggactctcgtgttgaagagattacctttgagagcttgaagagatgtgagtacttgaaagccgtgttgaacgaaacgttgagattacacccaagtgtcccaagaaacgcaagatttgcgattaaagacacgactttaccaagaggcggtggccccaacggcaaggatcctatcttgatcagaaagaatgaggtggtgcaatactccatctcggcaactcagacaaatcctgcttattatggcgccgatgctgctgattttagaccggaaagatggtttgagccatcaactagaaacttgggatgggcttacttgccattcaacggtggtccaagaatctgcttgggacaacagtttgctttgaccgaagccggttacgttttggttagacttgttcaggaattccctagcttgtcacaggaccccgaaactgagtacccaccacctagattggcacacttgacgatgtgcttgtttgacggggcatacgtcaagatgcaatag SEQ cytochrome P450atggctatatctagtttgctatcgtgggatgtgatctgtgtcgtcttcatttgcgtttgtgtttatttcgggtaID D2 (CYP52D2)tgaatattgttatactaaatacttgatgcacaaacatggcgctcgagaaatcgagaatgtgatcaacgatgggttcNO:  Nuc. Seqtttgggttccgcttacctttgctactcatgcgagccagcaatgagggccgacttatcgagttcagtgtcaag23 agattcgagtcggcgccacatccacagaacaagacattggtcaaccgggcattgagcgttcctgtgatactcaccaaggacccagtgaatatcaaagcgatgctatcgacccagtttgatgacttttcccttgggttgagactacaccagtttgcgccgttgttggggaaaggcatctttactttggacggcccagagtggaagcagagccgatctatgttgcgtccgcaatttgccaaagatcgggtttctcatatcctggatctagaaccgcattttgtgttgcttcggaagcacattgatggccacaatggagactacttcgacatccaggagctctacttccggttctcgatggatgtggcgacggggtttttgtttggcgagtctgtggggtcgttgaaagacgaagatgcgaggttcctggaagcattcaatgagtcgcagaagtatttggcaactagggcaacgttgcacgagttgtactttctttgtgacgggtttaggtttcgccagtacaacaaggttgtgcgaaagttctgcagccagtgtgtccacaaggcgttagatgttgcaccggaagacaccagcgagtacgtgtttctccgcgagttggtcaaacacactcgagatcccgttgttttacaagaccaagcgttgaacgtcttgcttgctggacgcgacaccaccgcgtcgttattatcgtttgcaacatttgagctagcccggaatgaccacatgtggaggaagctacgagaggaggttatcctgacgatgggaccgtccagtgatgaaataaccgtggccgggttgaagagttgccgttacctcaaagcaatcctaaacgaaactcttcgactatacccaagtgtgcctaggaacgcgagatttgctacgaggaatacgacgcttcctcgtggcggaggtccagatggatcgtttccgattttgataagaaagggccagccagtggggtatttcatttgtgctacacacttgaatgagaaggtatatgggaatgatagccatgtgtttcgaccggagagatgggctgcgttagagggcaagagtttgggctggtcgtatcttccattcaacggcggcccgagaagctgccttggtcagcagtttgcaatccttgaagcttcgtatgttttggctcgattgacacagtgctacacgacgatacagcttagaactaccgagtacccaccaaagaaactcgttcatctcacgatgagtcttctcaacggggtgtacatccgaactagaacttga SEQ cytochromeatgacaattaaagaaatgcctcagccaaaaacgtttggagagcttaaaaatttaccgttattaaacacagID P450: NADPHataaaccggttcaagctttgatgaaaattgcggatgaattaggagaaatctttaaattcgaggcgcctggtNO:  P450cgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatcacgctttgataaaa24 reductaseacttaagtcaagcgcttaaatttgtacgtgattttgcaggagacgggttatttacaagctggacgcatgaaa(Bacillusaaaattggaaaaaagcgcataatatcttacttccaagcttcagtcagcaggcaatgaaaggctatcatgmegaterium)cgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagcgtctaaatgcagatgagcatattganucleotideagtaccggaagacatgacacgtttaacgcttgatacaattggtctttgcggctttaactatcgctttaacagcNuc. Seqttttaccgagatcagcctcatccatttattacaagtatggtccgtgcactggatgaagcaatgaacaagctgcagcgagcaaatccagacgacccagcttatgatgaaaacaagcgccagtttcaagaagatatcaaggtgatgaacgacctagtagataaaattattgcagatcgcaaagcaagcggtgaacaaagcgatgatttattaacgcatatgctaaacggaaaagatccagaaacgggtgagccgcttgatgacgagaacattcgctatcaaattattacattcttaattgcgggacacgaaacaacaagtggtcttttatcatttgcgctgtatttcttagtgaaaaatccacatgtattacaaaaagcagcagaagaagcagcacgagttctagtagatcctgttccaagctacaaacaagtcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatggccaactgctcctgcgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaaaaaggcgacgaactaatggttctgattcctcagcttcaccgtgataaaacaatttggggagacgatgtggaagagttccgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaaacggtcagcgtgcgtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatgctaaaacactttgactttgaagatcatacaaactacgagctggatattaaagaaactttaacgttaaaacctgaaggctttgtggtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctagcactgaacagtctgctaaaaaagtacgcaaaaaggcagaaaacgctcataatacgccgctgcttgtgctatacggttcaaatatgggaacagctgaaggaacggcgcgtgatttagcagatattgcaatgagcaaaggatttgcaccgcaggtcgcaacgcttgattcacacgccggaaatcttccgcgcgaaggagctgtattaattgtaacggcgtcttataacggtcatccgcctgataacgcaaagcaatttgtcgactggttagaccaagcgtctgctgatgaagtaaaaggcgttcgctactccgtatttggatgcggcgataaaaactgggctactacgtatcaaaaagtgcctgcttttatcgatgaaacgcttgccgctaaaggggcagaaaacatcgctgaccgcggtgaagcagatgcaagcgacgactttgaaggcacatatgaagaatggcgtgaacatatgtggagtgacgtagcagcctactttaacctcgacattgaaaacagtgaagataataaatctactctttcacttcaatttgtcgacagcgccgcggatatgccgcttgcgaaaatgcacggtgcgttttcaacgaacgtcgtagcaagcaaagaacttcaacagccaggcagtgcacgaagcacgcgacatcttgaaattgaacttccaaaagaagcttcttatcaagaaggagatcatttaggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaaggttcggcctagatgcatcacagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccactcgctaaaacagtatccgtagaagagcttctgcaatacgtggagcttcaagatcctgttacgcgcacgcagcttcgcgcaatggctgctaaaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaaagcaagcctacaaagaacaagtgctggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccggcgtgtgaaatgaaattcagcgaatttatcgcccttctgccaagcatacgcccgcgctattactcgatttcttcatcacctcgtgtcgatgaaaaacaagcaagcatcacggtcagcgttgtctcaggagaagcgtggagcggatatggagaatataaaggaattgcgtcgaactatcttgccgagctgcaagaaggagatacgattacgtgctttatttccacaccgcagtcagaatttacgctgccaaaagaccctgaaacgccgcttatcatggtcggaccgggaacaggcgtcgcgccgtttagaggctttgtgcaggcgcgcaaacagctaaaagaacaaggacagtcacttggagaagcacatttatacttcggctgccgttcacctcatgaagactatctgtatcaagaagagcttgaaaacgcccaaagcgaaggcatcattacgcttcataccgctttttctcgcatgccaaatcagccgaaaacatacgttcagcacgtaatggaacaagacggcaagaaattgattgaacttcttgatcaaggagcgcacttctatatttgcggagacggaagccaaatggcacctgccgttgaagcaacgcttatgaaaagctatgctgacgttcaccaagtgagtgaagcagacgctcgcttatggctgcagcagctagaagaaaaaggccgatacgcaaaagacgtgtgggctgggtaa SEQ NADPHatggcattagataagttagatttatatgttattataacattggtggttgcaattgcagcttattttgcaaagaatcID cytochrome P450agtttcttgaccaacaacaagataccgggttccttaatactgatagtggagatggtaattcaagagatatctNO:  reductase, CPRtacaagctttgaagaagaacaataaaaatacgttattattatttggatcccaaacaggtacagcagaaga25 (Candida strainttatgccaacaaattgtcaagagaattgcattcaagatttggtttgaaaaccatggttgctgatttcgctgattATCC750)atgatttcgaaaacttcggagatattactgaagatatcttggttttctttattgttgctacttatggtgaaggtgaNuc. SeqaccaaccgataatgctgacgaatttcacacttggttgactgaagaagctgacaccttgagtactttgaaatatactgtttttggtttgggtaattcaacttatgaattcttcaatgctattggtagaaaatttgacagattgttgggagaaaaaggtggtgacagatttgctgaatacggtgaaggtgacgatggtactggtactttagatgaagatttcttggcctggaaggataacgtgtttgattccttaaagaatgatttgaattttgaagaaaaagagttgaaatacgaaccaaatgttaaattgactgaaagagatgatttatctggcaatgatccagatgtctccttgggtgaaccaaatgtcaaatacattaaatctgaaggtgttgacttaactaaaggtccatttgatcatactcatccatttttggctagaattgttaaaactaaagaattgtttacttctgaagacagacattgtgttcatgttgaatttgatatttctgaatcaaacttgaaatataccaccggtgatcatcttgcaatctggccatctaactctgatgaaaacattaagcaatttgccaaatgttttggtttagaagacaaacttgatactgttattgaattgaaagctttggattccacttattccatcccattccctaatccaatcacttatggagctgttattagacaccatttggaaatttcaggtcctgtttctagacaatttttcttatctattgctggatttgcccctgatgaagaaactaaaaagtcatttactagaattggtggtgataagcaagaatttgctagtaaagtcacccgtagaaaattcaacattgccgatgctttattatttgcttccaacaacagaccatggtccgatgttccattcgaattccttattgaaaatgtccaacacttaactcctcgttattactccatttcttcttcctcattaagtgaaaagcaaaccattaatgttactgctgttgttgaagccgaagaagaagctgatggaagaccagttactggtgttgtcaccaacttgttgaagaatattgaaattgaacaaaacaaaactggtgaaaccccaatggttcattatgatttgaatggtccaagaggcaaatttagcaagttcagattgccagttcacgttagaagatctaatttcaaattaccaaagaatagcactaccccagttattttgattggtccaggtaccggtgttgcaccattgagaggttttgttagagaaagagttcaacaagttaaaaatggtgttaatgttggtaagactgtattgttttatggatgtagaaattccgaacaagatttcttgtacaaacaagaatggagtgaatatgcctcagtattgggagaaaatttcgaaatgtttaatgccttctcaagacaagatccaactaagaaagtttatgttcaagataagattttagaaaatagtgctcttgttgatgagttattatctagtggagcaattatttatgtttgtggtgatgccagtagaatggctagagatgttcaagctgcaattgccaagattgttgccaaaagtagagatatccacgaagataaagctgctgaattggttaaatcttggaaagttcaaaatagataccaagaagatgtctggtaaSEQ NADPHatggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtagccgcctattttgctaagaaID cytochrome P450ccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaactccagagac NO: reductase A,gtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccagacgggtacggcaga26 CPRAagattacgccaacaaattgtccagagaattgcactccagatttggcttgaaaacgatggttgcagatttcg(Candida strainctgattacgattgggataacttcggagatatcaccgaagacatcttggtgtttttcattgttgccacctatggtATCC20336)gagggtgaacctaccgataatgccgacgagttccacacctggttgactgaagaagctgacactttgagtNuc. Seqaccttgaaatacaccgtgttcgggttgggtaactccacgtacgagttcttcaatgccattggtagaaagtttgacagattgttgagcgagaaaggtggtgacaggtttgctgaatacgctgaaggtgatgacggtactggcaccttggacgaagatttcatggcctggaaggacaatgtctttgacgccttgaagaatgatttgaactttgaagaaaaggaattgaagtacgaaccaaacgtgaaattgactgagagagacgacttgtctgctgctgactcccaagtttccttgggtgagccaaacaagaagtacatcaactccgagggcatcgacttgaccaagggtccattcgaccacacccacccatacttggccagaatcaccgagacgagagagttgttcagctccaaggacagacactgtatccacgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagctatctggccatccaactccgacgaaaacattaagcaatttgccaagtgtttcggattggaagataaactcgacactgttattgaattgaaggcgttggactccacttacaccatcccattcccaaccccaattacctacggtgctgtcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcaattgctgggtttgctcctgatgaagaaacaaagaaggcttttaccagacttggtggtgacaagcaagaattcgccgccaaggtcacccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacgctccatggtccgatgttccttttgaattccttattgaaaacgttccacacttgactccacgttactactccatttcgtcttcgtcattgagtgaaaagcaactcatcaacgttactgcagttgttgaagccgaagaagaagctgatggcagaccagtcactggtgttgtcaccaacttgttgaagaacgttgaaattgtgcaaaacaagactggcgaaaagccacttgtccactacgatttgagcggcccaagaggcaagttcaacaagttcaagttgccagtgcatgtgagaagatccaactttaagttgccaaagaactccaccaccccagttatcttgattggtccaggtactggtgttgccccattgagaggttttgtcagagaaagagttcaacaagtcaagaatggtgtcaatgttggcaagactttgttgttttatggttgcagaaactccaacgaggactttttgtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaactttgagatgttcaatgccttctccagacaagacccatccaagaaggtttacgtccaggataagattttagaaaacagccaacttgtgcacgagttgttgactgaaggtgccattatctacgtctgtggtgatgccagtagaatggctagagacgtgcagaccacaatttccaagattgttgctaaaagcagagaaattagtgaagacaaggctgctgaattggtcaagtcctggaaggtccaaaatagataccaagaagatgtttggtag SEQ NADPHatggctttagacaagttagatttgtatgtcatcataacattggtggtcgctgtggccgcctattttgctaagaaID cytochrome P450ccagttccttgatcagccccaggacaccgggttcctcaacacggacagcggaagcaactccagagac NO: reductase B,gtcttgctgacattgaagaagaataataaaaacacgttgttgttgtttgggtcccagaccggtacggcaga27 CPRBagattacgccaacaaattgtcaagagaattgcactccagatttggcttgaaaaccatggttgcagatttcg(Candida strainctgattacgattgggataacttcggagatatcaccgaagatatcttggtgtttttcatcgttgccacctacggtATCC20336)gagggtgaacctaccgacaatgccgacgagttccacacctggttgactgaagaagctgacactttgagtNuc. Seqactttgagatataccgtgttcgggttgggtaactccacctacgagttcttcaatgctattggtagaaagtttgacagattgttgagtgagaaaggtggtgacagatttgctgaatatgctgaaggtgacgacggcactggcaccttggacgaagatttcatggcctggaaggataatgtctttgacgccttgaagaatgacttgaactttgaagaaaaggaattgaagtacgaaccaaacgtgaaattgactgagagagatgacttgtctgctgccgactcccaagtttccttgggtgagccaaacaagaagtacatcaactccgagggcatcgacttgaccaagggtccattcgaccacacccacccatacttggccaggatcaccgagaccagagagttgttcagctccaaggaaagacactgtattcacgttgaatttgacatttctgaatcgaacttgaaatacaccaccggtgaccatctagccatctggccatccaactccgacgaaaacatcaagcaatttgccaagtgtttcggattggaagataaactcgacactgttattgaattgaaggcattggactccacttacaccattccattcccaactccaattacttacggtgctgtcattagacaccatttagaaatctccggtccagtctcgagacaattctttttgtcgattgctgggtttgctcctgatgaagaaacaaagaagactttcaccagacttggtggtgacaaacaagaattcgccaccaaggttacccgcagaaagttcaacattgccgatgccttgttatattcctccaacaacactccatggtccgatgttccttttgagttccttattgaaaacatccaacacttgactccacgttactactccatttcttcttcgtcgttgagtgaaaaacaactcatcaatgttactgcagtcgttgaggccgaagaagaagccgatggcagaccagtcactggtgttgttaccaacttgttgaagaacattgaaattgcgcaaaacaagactggcgaaaagccacttgttcactacgatttgagcggcccaagaggcaagttcaacaagttcaagttgccagtgcacgtgagaagatccaactttaagttgccaaagaactccaccaccccagttatcttgattggtccaggtactggtgttgccccattgagaggtttcgttagagaaagagttcaacaagtcaagaatggtgtcaatgttggcaagactttgttgttttatggttgcagaaactccaacgaggactttttgtacaagcaagaatgggccgagtacgcttctgttttgggtgaaaactttgagatgttcaatgccttctctagacaagacccatccaagaaggtttacgtccaggataagattttagaaaacagccaacttgtgcacgaattgttgaccgaaggtgccattatctacgtctgtggtgacgccagtagaatggccagagacgtccagaccacgatctccaagattgttgccaaaagcagagaaatcagtgaagacaaggccgctgaattggtcaagtcctggaaagtccaaaatagataccaagaagatgtttggtag SEQ cytochromemtikempqpktfgelknlpllntdkpvqalmkiadelgeifkfeapgrvtrylssqrlikeacdesrfdknlsqID P450: NADPHalkfvrdfagdglftswtheknwkkahnillpsfsqqamkgyhammvdiavqlvqkwerlnadehievNO:  P450 reductasepedmtrltldtiglcgfnyrfnsfyrdqphpfitsmvrasdeamnksqranpddpaydenkrqfqedikv28 (Bacillusmndlvdkiiadrkasgeqsddllthmlngkdpetgeplddeniryqiitfliaghettsgllsfasyflvknphmegaterium)vlqkaaeeaarylvdpvpsykqvkqlkyvgmvlneasrlwptapafslyakedtvlggeyplekgdelmamino acidvsipqlhrdktiwgddveefrperfenpsaipqhafkpfgngqracigqqfalheatsvlgmmlkhfdfed[P450 activityhtnyesdiketltlkpegfvvkakskkiplggipspsteqsakkvrkkaenahntpslvlygsnmgtaegtshown inardladiamskgfapqvatldshagnlpregavlivtasynghppdnakqfvdwldqasadevkgvryitalics,svfgcgdknwattyqkvpafidetlaakgaeniadrgeadasddlegtyeewrehmwsdvaayfnldiP450 reductaseensednkstlslqfvdsaadmplakmhgafstnvvaskelqqpgsarstrhleielpkeasyqegdhlgactivity shownviprnyegivnrvtarfgldasqqirseaeeeklahlplaktvsveelsqyvelqdpvtrtqlramaaktvcpinphkveleallekqaykeqvsakrltmleslekypacemkfsefialspsirpryysisssprvdekqasitvnormal font]svvsgeawsgygeykgiasnylaesqegdtitcfistpqseftspkdpetplimvgpgtgvapfrgfvqarA.A. Seqkqlkeqgqslgeahlyfgcrsphedysyqeelenaqsegiitlhtafsrmpnqpktyvqhvmeqdgkklielldqgahfyicgdgsqmapaveatlmksyadvhqvseadarlwsqqleekgryakdvwag* SEQacyl CoA ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAGGTCCTGACCCTA ID oxidase,GATCATCCATCCAAAAGGAAAGAGACAGCTCCAAATGGAACCCTCAAC NO:  PDX4 (CandidaAAATGAACTACTTCTTGGAAGGCTCCGTCGAAAGAAGTGAGTTGATGA 29 strainAGGCTTTGGCCCAACAAATGGAAAGAGACCCAATCTTGTTCACAGACG ATCC20336)GCTCCTACTACGACTTGACCAAGGACCAACAAAGAGAATTGACCGCCG nucleotideTCAAGATCAACAGAATCGCCAGATACAGAGAACAAGAATCCATCGACACTTTCAACAAGAGATTGTCCTTGATTGGTATCTTTGACCCACAGGTCGGTACCAGAATTGGTGTCAACCTCGGTTTGTTCCTTTCTTGTATCAGAGGTAACGGTACCACTTCCCAATTGAACTACTGGGCTAACGAAAAGGAAACCGCTGACGTTAAAGGTATCTACGGTTGTTTCGGTATGACCGAATTGGCCCACGGTTCCAACGTTGCTGGTTTGGAAACCACCGCCACATTTGACAAGGAATCTGACGAGTTTGTCATCAACACCCCACACATTGGTGCCACCAAGTGGTGGATTGGTGGTGCTGCTCACTCCGCCACCCACTGTTCTGTCTACGCCAGATTGATTGTTGACGGTCAAGATTACGGTGTCAAGACTTTTGTTGTCCCATTGAGAGACTCCAACCACGACCTCATGCCAGGTGTCACTGTTGGTGACATTGGTGCCAAGATGGGTAGAGATGGTATCGATAACGGTTGGATCCAATTCTCCAACGTCAGAATCCCAAGATTCTTTATGTTGCAAAAGTTCTGTAAGGTTTCTGCTGAAGGTGAAGTCACCTTGCCACCTTTGGAACAATTGTCTTACTCCGCCTTGTTGGGTGGTAGAGTCATGATGGTTTTGGACTCCTACAGAATGTTGGCTAGAATGTCCACCATTGCCTTGAGATACGCCATTGGTAGAAGACAATTCAAGGGTGACAATGTCGATCCAAAAGATCCAAACGCTTTGGAAACCCAATTGATAGATTACCCATTGCACCAAAAGAGATTGTTCCCATACTTGGCTGCTGCCTACGTCATCTCCGCTGGTGCCCTCAAGGTTGAAGACACCATCCATAACACCTTGGCTGAATTGGACGCTGCCGTTGAAAAGAACGACACCAAGGCTATCTTTAAGTCTATTGACGACATGAAGTCATTGTTTGTTGACTCTGGTTCCTTGAAGTCCACTGCCACTTGGTTGGGTGCTGAAGCCATTGACCAATGTAGACAAGCCTGTGGTGGTCACGGTTACTCGTCCTACAACGGCTTCGGTAAAGCCTACAACGATTGGGTTGTCCAATGTACTTGGGAAGGTGACAACAATGTCTTGGCCATGAGTGTTGGTAAGCCAATTGTCAAGCAAGTTATCAGCATTGAAGATGCCGGCAAGACCGTCAGAGGTTCCACCGCTTTCTTGAACCAATTGAAGGACTACACTGGTTCCAACAGCTCCAAGGTTGTTTTGAACACTGTTGCTGACTTGGACGACATCAAGACTGTCATCAAGGCTATTGAAGTTGCCATCATCAGATTGTCCCAAGAAGCTGCTTCTATTGTCAAGAAGGAATCTTTCGACTATGTCGGCGCTGAATTGGTTCAACTCTCCAAGTTGAAGGCTCACCACTACTTGTTGACTGAATACATCAGAAGAATTGACACCTTTGACCAAAAGGACTTGGTTCCATACTTGATCACCCTCGGTAAGTTGTACGCTGCCACTATTGTCTTGGACAGATTTGCCGGTGTCTTCTTGACTTTCAACGTTGCCTCCACCGAAGCCATCACTGCTTTGGCCTCTGTGCAAATTCCAAAGTTGTGTGCTGAAGTCAGACCAAACGTTGTTGCTTACACCGACTCCTTCCAACAATCCGACATGATTGTCAATTCTGCTATTGGTAGATACGATGGTGACATCTATGAGAACTACTTTGACTTGGTCAAGTTGCAGAACCCACCATCCAAGACCAAGGCTCCTTACTCTGATGCTTTGGAAGCCATGTTGAACAGACCAACCTTGGACGAAAGAGAAAGATTTGAAAAGTCTGATGAAACCGCTGCTATCTTGT CCAAGTAA SEQ acyl CoAMTFTKKNVSVSQGPDPRSSIQKERDSSKWNPQQMNYFLEGSVERSELM ID oxidase,KALAQQMERDPILFTDGSYYDLTKDQQRELTAVKINRIARYREQESIDTFN NO:  PDX4 (CandidaKRLSLIGIFDPQVGTRIGVNLGLFLSCIRGNGTTSQLNYWANEKETADVKGI 30 strainYGCFGMTELAHGSNVAGLETTATFDKESDEFVINTPHIGATKWWIGGAAH ATCC20336)SATHCSVYARLIVDGQDYGVKTFVVPLRDSNHDLMPGVTVGDIGAKMGR amino acidDGIDNGWIQFSNVRIPRFFMLQKFCKVSAEGEVTLPPLEQLSYSALLGGRVMMVLDSYRMLARMSTIALRYAIGRRQFKGDNVDPKDPNALETQLIDYPLHQKRLFPYLAAAYVISAGALKVEDTIHNTLAELDAAVEKNDTKAIFKSIDDMKSLFVDSGSLKSTATWLGAEAIDQCRQACGGHGYSSYNGFGKAYNDWVVQCTWEGDNNVLAMSVGKPIVKQVISIEDAGKTVRGSTAFLNQLKDYTGSNSSKVVLNTVADLDDIKTVIKAIEVAIIRLSQEAASIVKKESFDYVGAELVQLSKLKAHHYLLTEYIRRIDTFDQKDLVPYLITLGKLYAATIVLDRFAGVFLTFNVASTEAITALASVQIPKLCAEVRPNVVAYTDSFQQSDMIVNSAIGRYDGDIYENYFDLVKLQNPPSKTKAPYSDALEAMLNRPTLDERERFEKSDETAAILSK * SEQ acyl CoAATGCCTACCGAACTTCAAAAAGAAAGAGAACTCACCAAGTTCAACCCA ID oxidase,AAGGAGTTGAACTACTTCTTGGAAGGTTCCCAAGAAAGATCCGAGATC NO:  PDX5 (CandidaATCAGCAACATGGTCGAACAAATGCAAAAAGACCCTATCTTGAAGGTC 31 strainGACGCTTCATACTACAACTTGACCAAAGACCAACAAAGAGAAGTCACC ATCC20336)GCCAAGAAGATTGCCAGACTCTCCAGATACTTTGAGCACGAGTACCCA nucleotideGACCAACAGGCCCAGAGATTGTCGATCCTCGGTGTCTTTGACCCACAAGTCTTCACCAGAATCGGTGTCAACTTGGGTTTGTTTGTTTCCTGTGTCCGTGGTAACGGTACCAACTCCCAGTTCTTCTACTGGACCATAAATAAGGGTATCGACAAGTTGAGAGGTATCTATGGTTGTTTTGGTATGACTGAGTTGGCCCACGGTTCCAACGTCCAAGGTATTGAAACCACCGCCACTTTTGACGAAGACACTGACGAGTTTGTCATCAACACCCCACACATTGGTGCCACCAAGTGGTGGATCGGTGGTGCTGCGCACTCCGCCACCCACTGCTCCGTCTACGCCAGATTGAAGGTCAAAGGAAAGGACTACGGTGTCAAGACCTTTGTTGTCCCATTGAGAGACTCCAACCACGACCTCGAGCCAGGTGTGACTGTTGGTGACATTGGTGCCAAGATGGGTAGAGACGGTATCGATAACGGTTGGATCCAGTTCTCCAACGTCAGAATCCCAAGATTCTTTATGTTGCAAAAGTACTGTAAGGTTTCCCGTCTGGGTGAAGTCACCATGCCACCATCTGAACAATTGTCTTACTCGGCTTTGATTGGTGGTAGAGTCACCATGATGATGGACTCCTACAGAATGACCAGTAGATTCATCACCATTGCCTTGAGATACGCCATCCACAGAAGACAATTCAAGAAGAAGGACACCGATACCATTGAAACCAAGTTGATTGACTACCCATTGCATCAAAAGAGATTGTTCCCATTCTTGGCTGCCGCTTACTTGTTCTCCCAAGGTGCCTTGTACTTAGAACAAACCATGAACGCAACCAACGACAAGTTGGACGAAGCTGTCAGTGCTGGTGAAAAGGAAGCCATTGACGCTGCCATTGTCGAATCCAAGAAATTGTTCGTCGCTTCCGGTTGTTTGAAGTCCACCTGTACCTGGTTGACTGCTGAAGCCATTGACGAAGCTCGTCAAGCTTGTGGTGGTCACGGTTACTCGTCTTACAACGGTTTCGGTAAAGCCTACTCCGACTGGGTTGTCCAATGTACCTGGGAAGGTGACAACAACATCTTGGCCATGAACGTTGCCAAGCCAATGGTTAGAGACTTGTTGAAGGAGCCAGAACAAAAGGGATTGGTTCTCTCCAGCGTTGCCGACTTGGACGACCCAGCCAAGTTGGTTAAGGCTTTCGACCACGCCCTTTCCGGCTTGGCCAGAGACATTGGTGCTGTTGCTGAAGACAAGGGTTTCGACATTACCGGTCCAAGTTTGGTTTTGGTTTCCAAGTTGAACGCTCACAGATTCTTGATTGACGGTTTCTTCAAGCGTATCACCCCAGAATGGTCTGAAGTCTTGAGACCTTTGGGTTTCTTGTATGCCGACTGGATCTTGACCAACTTTGGTGCCACCTTCTTGCAGTACGGTATCATTACCCCAGATGTCAGCAGAAAGATTTCCTCCGAGCACTTCCCAGCCTTGTGTGCCAAGGTTAGACCAAACGTTGTTGGTTTGACTGATGGTTTCAACTTGACTGACATGATGACCAATGCTGCTATTGGTAGATATGATGGTAACGTCTACGAACACTACTTCGAAACTGTCAAGGCTTTGAACCCACCAGAAAACACCAAGGCTCCATACTCCAAGGCTTTGGAAGACATGTTGAACCGTCCAGACCTTGAAGTCAGAGAAAGAGGTGAAAAGTCCGAAGAAGCTGCTG AAATCTTGTCCAGTTAA SEQacyl CoA MPTELQKERELTKFNPKELNYFLEGSQERSEIISNMVEQMQKDPILKVDAS ID oxidase,YYNLTKDQQREVTAKKIARLSRYFEHEYPDQQAQRLSILGVFDPQVFTRIG NO:  PDX5 (CandidaVNLGLFVSCVRGNGTNSQFFYWTINKGIDKLRGIYGCFGMTELAHGSNVQ 32 strainGIETTATFDEDTDEFVINTPHIGATKWWIGGAAHSATHCSVYARLKVKGKD ATCC20336)YGVKTFVVPLRDSNHDLEPGVTVGDIGAKMGRDGIDNGWIQFSNVRIPRF amino acidFMLQKYCKVSRSGEVTMPPSEQLSYSALIGGRVTMMMDSYRMTSRFITIALRYAIHRRQFKKKDTDTIETKLIDYPLHQKRLFPFLAAAYLFSQGALYLEQTMNATNDKLDEAVSAGEKEAIDAAIVESKKLFVASGCLKSTCTWLTAEAIDEARQACGGHGYSSYNGFGKAYSDWVVQCTWEGDNNILAMNVAKPMVRDLLKEPEQKGLVLSSVADLDDPAKLVKAFDHALSGLARDIGAVAEDKGFDITGPSLVLVSKLNAHRFLIDGFFKRITPEWSEVLRPLGFLYADWILTNFGATFLQYGIITPDVSRKISSEHFPALCAKVRPNVVGLTDGFNLTDMMTNAAIGRYDGNVYEHYFETVKALNPPENTKAPYSKALEDMLNRPDLEVRERGEKSEEA AEILSS* SEQ Acyl-CoAatgatcagaaccgtccgttatcaatccctcaagaggttcagacctctggctttgtctcctgtttttcgtccacgID Hydrolase (ACHA)ctacaactcccagaaggccaatttccaccgtccagaccaccctgggtccgacgagccagctgaagcc NO: Nucleotide Seqgccgacgccgccgccacgatcctcgccgagttgcgagacaagcagacgaacccgaacaaggccac 33ctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaagacgattgacagtttcagctacgttgaccccaagacgaccgtcgtgggggagaagacacgcagtgactcgttctcgttcttgttgttgccgttcaaggacgacaagtggttgtgtgacgcgtacatcaatgcgtttggccggttgcgtgtagcgcagttgttccaggacttggacgccttggcggggcgcatcgcgtacaggcactgttccccagcggagcccgtgaatgtcacggcgagcgtggatagggtgtacatggtgaagaaagtggacgagattaacaattacaatttcgtgttggcggggtccgtgacgtggaccgggagatcgtcgatggagatcacggtgaaagggtatgcttttgaagacgccgtgccggatataacgaacgaggagtccttgccggcagagaatgtgtttttggctgctaatttcaccttcgtggcacggaacccacttacacacaagtcctttgctattaacagattgttgcccgtgactgagaaggactgggtcgactatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaagatcttggagcctaccgcggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacagaacatcgagagggccaacgatgggatcgcgttcatgaaggacacgaccatgaagtccaccttgttcatgcagccccagtaccgtaacagacactcatacatgattttcggagggtacttgttaagacaaactttcgaattggcctactgtaccgcggcaacgttttccctggccgggccccgtttcgtcagcttggactccaccacgttcaagaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatctcgtacacggagcacgtgcacgagggagtggaggagattgacgcggactcaccgttcaacttcagcttgcctgccacgaacaagatctcgaagaaccccgaggcgttcttgtcggaacccggcacgttgattcaagtcaaggtcgacacatacatccaggagttagagcagagtgtgaagaagcccgcgggtacgttcatctactcgttctatgttgataaagaaagcgttactgttgatggaaaggcgtcgttttgttcagttatcccgcagacgtactccgagatgatgacttatgtgggcgggagaagaagagcccaggatactgctaactacgtggagactttgccgtttagtggaagcggcaat taaSEQ Acyl-CoA MIRTVRYQSLKRFRPSALSPVFRPRYNSQKANFHRPDHPGSDEPAEAAD IDHydrolase (ACHA) AAATILAELRDKQTNPNKATWLDALTEREKLRAEGKTIDSFSYVDPKTTVVNO:  Amino Acid Seq GEKTRSDSFSFLLLPFKDDKWLCDAYINAFGRLRVAQLFQDLDALAGRIAY34 RHCSPAEPVNVTASVDRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEITVKGYAFEDAVPDITNEESLPAENVFLAANFTFVARNPLTHKSFAINRLLPVTEKDWVDYRRAESHNAKKKLMAKNKKILEPTAEESKLIYDMWRSSKSLQNIERANDGIAFMKDTTMKSTLFMQPQYRNRHSYMIFGGYLLRQTFELAYCTAATFSSAGPRFVSLDSTTFKNPVPVGSVLTMDSSISYTEHVHEGVEEIDADSPFNFSLPATNKISKNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYSFYVDKESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVETLPFSG SGN SEQ Acyl-CoAatgatcagaaccgtccgttatcaatccttcaagaggttcaaacctctgactttatcccccgttttccgtccacID Hydrolase (ACHB)gctacaactcccagaaggccaatttccaccgtccagaccacgctgggtccgacgagccagccgaagc NO: Nucleotide Seqcgccgacgccgctgccacgatcctcgccgagttgcgagacaagcagacgaacccgaacaaggcca 35cctggctcgatgcgttaacggagcgggagaagttgcgtgccgagggcaagacaatcgacagcttcagctacgttgaccccaagacaaccgtcgtgggggagaagacacgcagcgactcgttctcgttcttgttgttgccgttcaaggacgacaagtggttgtgtgacgcgtacatcaatgcgtttggccggttgcgtgtagcgcagttgttccaggacttggacgccttggcgggccgcatcgcgtacaggcactgttcccccgctgagcccgtgaatgtcacggcgagcgtggatagagtgtatatggtgaagaaagtggacgagattaataattacaatttcgtgttggcggggtccgtgacgtggaccgggagatcgtcgatggagatcacggtcaaagggtatgcttttgaagacgccgtgccggagataactaacgaggagtccttgccggcagagaatgtgttcttggctgttaatttcaccttcgtggcacgtaacccactcacacacaagtccttcgctattaacagattgttgcccgtgactgagaaggactgggtcgattatcgccgtgctgagtcccacaacgccaagaagaagttgatggcaaagaacaagaagatcttggagcctaccccggaagagtccaagttgatctacgacatgtggagatcgtccaagtccttacagaacatcgagaaggccaacgacgggatcgcgttcatgaaggacacgataatgaagtccaccttgttcatgcagccccagtaccgtaacagacactcatacatgattttcggtgggtatttgttaagacaaactttcgaattggcctattgtaccgcagcaacgttttccctggcgggaccccgtttcgtcagcttggactccaccacgttcaagaaccccgtgcccgtggggtcggtgctcaccatggactcgtcgatctcgtacacggagcacgtccacgatggcgttgaggagattgacgccgactccccgttcaacttcagcttgcctgccacgaacaagatctcgaagaaccccgaggcgttcttgtcggagcccggcacgttgatccaagtcaaggtcgacacgtacatccaggagttagagcaaagtgtgaagaagcctgcgggaacgttcatctactcgttctatgttgataaagagagcgttactgtggatggaaaggcgtcgttttgttcagttatcccgcagacgtactccgagatgatgacttatgtgggcgggagaagaagagcccaggatactgctaattacgtggagactttgccgtttagtggaagcggcaattaa SEQAcyl-CoA MIRTVRYQSFKRFKPLTLSPVFRPRYNSQKANFHRPDHAGSDEPAEAADA IDHydrolase (ACHB) AATILAELRDKQTNPNKATWLDALTEREKLRAEGKTIDSFSYVDPKTTVVGNO:  EKTRSDSFSFLLLPFKDDKWLCDAYINAFGRLRVAQLFQDLDALAGRIAYR 36HCSPAEPVNVTASVDRVYMVKKVDEINNYNFVLAGSVTWTGRSSMEITVKGYAFEDAVPEITNEESLPAENVFLAVNFTFVARNPLTHKSFAINRLLPVTEKDWVDYRRAESHNAKKKLMAKNKKILEPTPEESKLIYDMWRSSKSLQNIEKANDGIAFMKDTIMKSTLFMQPQYRNRHSYMIFGGYLLRQTFELAYCTAATFSLAGPRFVSLDSTTFKNPVPVGSVLTMDSSISYTEHVHDGVEEIDADSPFNFSLPATNKISKNPEAFLSEPGTLIQVKVDTYIQELEQSVKKPAGTFIYSFYVDKESVTVDGKASFCSVIPQTYSEMMTYVGGRRRAQDTANYVETLPFSG SGN SEQE. coli Acyl-CoAatggccgatacattgctcatcttgggtgactctttgtctgcagggtatcggatgtccgcatctgccgcatggcID ThioesterasectgcactcctcaatgacaaatggcaaagcaagacatcggtcgtgaatgcatctatctctggcgatacctcNO:  (TESA) genegcagcaggggttggcccgtctcccagccttgttgaagcaacatcaaccacgttgggtcttggtcgaattg37 without signalggcggcaatgatggtctcagaggttttcaacctcaacagaccgagcagacattgcgtcaaatcctccaapeptide sequencegacgtgaaggcagcaaacgccgaacctctcttgatgcagataagattgcctgccaactatggtcgtagaoptimized for C.tacaatgaagccttttctgcaatctacccgaagcttgcaaaggagtttgacgtcccattgttgccgtttttgattropicalisggaagaggtgtaccttaagcctcagtggatgcaagacgatggtatccatccgaaccgtgatgcacaacNucleotide Seqcattcatcgcagattggatggccaaacaactccaacctttggtcaatcatgatagctaa SEQE. coli Acyl-CoA MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKTSVVNASISGDTSQ IDThioesterase QGLARLPALLKQHQPRWVLVELGGNDGLRGFQPQQTEQTLRQILQDVKA NO: (TESA) without ANAEPLLMQIRLPANYGRRYNEAFSAIYPKLAKEFDVPLLPFLMEEVYLKP 38signal peptide QWMQDDGIHPNRDAQPFIADWMAKQLQPLVNHDS Amino Acid Seq SEQAcyl-CoAatgggtgcccctttaacagtcgccgttggcgaagcaaaaccaggcgaaaccgctccaagaagaaaa IDSynthetasegccgctcaaaaaatggcctctgtcgaacgcccaacagactcaaaggcaaccactttgccagacttcattNO:  (ACS1)gaagagtgttttgccagaaacggcaccagagatgccatggcctggagagacttggtcgaaatccacgt 39Nuc. Seqcgaaaccaaacaggttaccaaaatcattgacggcgaacagaaaaaggtcgataaggactggatctactacgaaatgggtccttacaactacatatcctaccccaagttgttgacgttggtcaagaactactccaagggtttgttggagttgggcttggccccagatcaagaatccaagttgatgatctttgccagtacctcccacaagtggatgcagaccttcttagcctccagtttccaaggtatccccgttgtcaccgcctacgacaccttgggtgagtcgggcttgacccactccttggtgcaaaccgaatccgatgccgtgttcaccgacaaccaattgttgtcctccttgattcgtcctttggagaaggccacctccgtcaagtatgtcatccacggggaaaagattgaccctaacgacaagagacagggcggcaaaatctaccaggatgcggaaaaggccaaggagaagattttacaaattagaccagatattaaatttatttctttcgacgaggttgttgcattgggtgaacaatcgtccaaagaattgcatttcccaaaaccagaagacccaatctgtatcatgtacacctcgggttccaccggtgctccaaagggtgtggttatcaccaatgccaacattgttgccgccgtgggtggtatctccaccaatgctactagagacttggttagaactgtcgacagagtgattgcatttttgccattggcccacattttcgagttggcctttgagttggttaccttctggtggggggctccattgggttacgccaatgtcaagactttgaccgaagcctcctgcagaaactgtcagccagacttgattgaattcaaaccaaccatcatggttggtgttgctgccgtttgggaatcggtcagaaagggtgtcttgtctaaattgaaacaggcttctccaatccaacaaaagatcttctgggctgcattcaatgccaagtctactttgaaccgttatggcttgccaggcggtgggttgtttgacgctgtcttcaagaaggttaaagccgccactggtggccaattgcgttatgtgttgaatggtgggtccccaatctctgttgatgcccaagtgtttatctccaccttgcttgcgccaatgttgttgggttacggtttgactgaaacctgtgccaataccaccattgtcgaacacacgcgcttccagattggtactttgggtaccttggttggatctgtcactgccaagttggttgatgttgctgatgctggatactacgccaagaacaaccagggtgaaatctggttgaaaggcggtccagttgtcaaggaatactacaagaacgaagaagaaaccaaggctgcattcaccgaagatggctggttcaagactggtgatattggtgaatggaccgccgacggtggtttgaacatcattgaccgtaagaagaacttggtcaagactttgaatggtgaatacattgctttggagaaattggaaagtatttacagatccaaccacttgattttgaacttgtgtgtttacgctgaccaaaccaaggtcaagccaattgctattgtcttgccaattgaagccaacttgaagtctatgttgaaggacgaaaagattatcccagatgctgattcacaagaattgagcagcttggttcacaacaagaaggttgcccaagctgtcttgagacacttgctccaaaccggtaaacaacaaggtttgaaaggtattgaattgttgcagaatgttgtcttgttggatgacgagtggaccccacagaatggttttgttacttctgcccaaaagttgcagagaaagaagattttagaaagttgtaaaaaagaagttgaagaggcatacaagtcgtcttag SEQ Acyl-CoAMGAPLTVAVGEAKPGETAPRRKAAQKMASVERPTDSKATTLPDFIEECFA ID SynthetaseRNGTRDAMAWRDLVEIHVETKQVTKIIDGEQKKVDKDWIYYEMGPYNYIS NO:  (ACS1)YPKLLTLVKNYSKGLLELGLAPDQESKLMIFASTSHKWMQTFLASSFQGIP 40 A.A. SeqVVTAYDTLGESGLTHSLVQTESDAVFTDNQLLSSLIRPLEKATSVKYVIHGEKIDPNDKRQGGKIYQDAEKAKEKILQIRPDIKFISFDEVVALGEQSSKELHFPKPEDPICIMYTSGSTGAPKGVVITNANIVAAVGGISTNATRDLVRTVDRVIAFLPLAHIFELAFELVTFWWGAPLGYANVKTLTEASCRNCQPDLIEFKPTIMVGVAAVWESVRKGVLSKLKQASPIQQKIFWAAFNAKSTLNRYGLPGGGLFDAVFKKVKAATGGQLRYVLNGGSPISVDAQVFISTLLAPMLLGYGLTETCANTTIVEHTRFQIGTLGTLVGSVTAKLVDVADAGYYAKNNQGEIWLKGGPVVKEYYKNEEETKAAFTEDGWFKTGDIGEWTADGGLNIIDRKKNLVKTLNGEYIALEKLESIYRSNHLILNLCVYADQTKVKPIAIVLPIEANLKSMLKDEKIIPDADSQELSSLVHNKKVAQAVLRHLLQTGKQQGLKGIELLQNVVLLDDEWTPQNGFVTSAQKLQRKKILESCKKEVEEAYKSS SEQ Long-chain Acyl-atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattgccID CoA SynthetasegacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccagcagaggtNO:  (FAT1)aaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaagcgttggcgttcc41 Nuc. Seqcaagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgacgatcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttgaagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattgtcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagccattgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcagagataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttgtttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaaccgatactgactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcatgtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagcagaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctgtaccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcagaaatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaaaggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcgacagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattgatgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggtaaagccaaattg SEQ Long-chainMSGLEIAAAAILGSQLLEAKYLIADDVSLAKTVAVNALPYLWKASRGKASY ID Acyl-CoA WYFFEQSVFKNPNNKALAFPRPRKNAPTPKTDAEGFQIYDDQFDLEEYTY NO:  SynthetaseKELYDMVLKYSYILKNEYGVTANDTIGVSCMNKPLFIVLWLALWNIGALPAF 42 (FAT1)LNFNTKDKPLIHCLKIVNASQVFVDPDCDSPIRDTEAQIREELPHVQINYIDE A.A. SeqFALFDRLRLKSTPKHRAEDKTRRPTDTDSSACALIYTSGTTGLPKAGIMSWRKAFMASVFFGHIMKIDSKSNVLTAMPLYHSTAAMLGLCPTLIVGGCVSVSQKFSATSFWTQARLCGATHVQYVGEVCRYLLNSKPHPDQDRHNVRIAYGNGLRPDIWSEFKRRFHIEGIGEFYAATESPIATTNLQYGEYGVGACRKYGSLISLLLSTQQKLAKMDPEDESEIYKDPKTGFCTEAAYNEPGELLMRILNPNDVQKSFQGYYGNKSATNSKILTNVFKKGDAWYRSGDLLKMDEDKLLYFVDRLGDTFRWKSENVSATEVENELMGSKALKQSVVVGVKVPNHEGRACFAVCEAKDELSHEEILKLIHSHVTKSLPVYAQPAFIKIGTIEASHNHKVPKNQFKNQKLPKGEDGKDLIYWLNGDKYQELTEDDWSLICTGKAKL SEQ Acyl-CoAatgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatccacgtcttcgttccID Sterolacyl aggaagaactagtcgaggttgaaatgtccaactcgtccattaacgaatcccagaccgatgagtcgtaccNO:  transferasegtcctgaagaaacctcattgcattacaggaggaagtcccacaggaccccgtcagaggagtcgttccta 43(ARE1)gagatcaccaagaacgtgaatgatccggatctagtttccaagattgagaacctaaggggcaaagtaagNuc. Seqccaacgggaagacaggttgaggaagcactaccttcacacctcccaggacgtcaagttcttgtcccggttcaacgacatcaagttcaagctgaactccgcgacgattctagattcggatgcgttttacaagagtgaatactttggagtcttgaccatcttctgggtggttatcgcactctacatattgtcaacgttgtcagatgtttactttggcatggccaagcccttactggactggatcatcataggaatgttcaagcaggacttggtgaaagttgcactcgttgatcttgccatgtacctatcctcgtattttccttatttcttgcaggttgcatgcaaacggggtgatgtatcttggcatggtcttggatgggcaatacagggggtttacagcttggtgtttttgacgttctggacggtagttccgcaggagttggccatggatcttccttggattgcacgaattttcttgatcttgcattgcttggtgtttattatgaagatgcagtcgtatgggcattacaatggatacctttgggatgtgtatcaggaaggattggcctctgaggctgatctcagggacctttctgagtatgatgaagatttccccctggatcacgtggaggttctagaacagagcttgtggtttgccaaacacgagttggagtttcaatcgaatggaactgctgagaggaaggaccaccatcaccatgtattcgacgaaaaggatgtcaacaaaccaatacgtgtcttgcaagaagagggaattatcaagtttccggcaaacatcaacttcaaggattatttcgagtacagtatgttcccaacgctagtctacacgttgagcttcccccgaactcgacagattagatggacgtatgtgttgcagaaggttttgggaacatttgccttagtgtttgccatgattatcgtcgccgaagagagtttctgccccttgatgcaagaagttgatcagtacacaaaattgccaaccaaccaaaggttcccaaaatacttcgtcgttctttcccacttgatattaccgctcggcaagcagtacttgctctcattcatcctcatctggaatgaaattctcaacggcatagcggagttaagcaggtttggcgaccggcatttctacggcgcttggtggtcgagcgtcgattacatggactattcaagaaaatggaacaccatcgtgcaccgattcctccgtcggcacgtttacaattcgagcattcacatcctcggtatttccaggacgcaagccgcgatagttacacttttgctttctgccacaatccacgaactcgttatgtacgtcctatttggcaaattacgagggtacctattccttacgatgcttgtccagatccccatgaccgtcacctccaagttcaacaaccgtgtttggggcaacatcatgttctggttgacgtatttatctggccccagcttggttagtgcgttgtatttactcttctag SEQ Acyl-CoAMSDDEIAGIVIEIDDDVKSTSSFQEELVEVEMSNSSINESQTDESYRPEETS ID SterolacylLHYRRKSHRTPSEESFLEITKNVNDPDLVSKIENLRGKVSQREDRLRKHYL NO:  transferaseHTSQDVKFLSRFNDIKFKLNSATILDSDAFYKSEYFGVLTIFWVVIALYILST 44 (ARE1)LSDVYFGMAKPLLDWIIIGMFKQDLVKVALVDLAMYLSSYFPYFLQVACKR A.A. SeqGDVSWHGLGWAIQGVYSLVFLTFWTVVPQELAMDLPWIARIFLILHCLVFIMKMQSYGHYNGYLWDVYQEGLASEADLRDLSEYDEDFPLDHVEVLEQSLWFAKHELEFQSNGTAERKDHHHHVFDEKDVNKPIRVLQEEGIIKFPANINFKDYFEYSMFPTLVYTLSFPRTRQIRWTYVLQKVLGTFALVFAMIIVAEESFCPLMQEVDQYTKLPTNQRFPKYFVVLSHLILPLGKQYLLSFILIWNEILNGIAELSRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHVYNSSIHILGISRTQAAIVTLLLSATIHELVMYVLFGKLRGYLFLTMLVQIPMTVTSKFNNRVWGNIMFWLTYLSGPSLVSALYLLF SEQ Acyl-CoAatgtccgacgacgagatagcaggaatagtcattgaaatcgacgatgacgtgaaatctacgtcttcgttccID SterolacylaggaagacctagtcgaggttgagatgtccaactcgtccattaacgaatcccagacggatgagttgtcgtaNO:  transferaseccgtcctgaagaaatctcattgcattcgagaaggaagtcccacaagaccccgtcagatgagtcgttccta45 (ARE2)gagatcaccaagaacgtgaatgatccggatctagtctccaagattgagaacttaaggggcaaagtaagNuc. Seqccaacgggaagacaggttgaggaaacactacctccacacatcccaggacgtcaagttcttgtctcggttcaacgacatcaagttcaagctgaactccgcgacgattctagattcggatgcgttttacaagagcgagcactttggagtcttgactatcttctgggtggttatcggactctacataatgtcaacgttgtcagacatgtattttggcatggccaagcccttactggactggataatcataggaatgttcaagaaggatttgatgcaagttgcactcgttgatcttgtcatgtacttatcctcgtattttccttatttcctacaggttgcatgcaagaccggagctatatcttggcatggtcttggatgggccatacagggggtttacagcttggtgtttttaactttctgggcggtacttccgctggagctggccatggatcttccttggattgcacgagttttcttgatcttgcattgcttggtgtttattatgaagatgcaatcatatggacattacaatggatacctttgggatgtatatcaggaaggattggtctcggaagctgatctcacggctgtttctgagtatgatgatgatttccccctggatcacggggaggttctagaacagagcttgtggttcgccaaacacgagttggagtttcaatctaatggaactacggagaggaaggatcaccatcatcatgtattcgacgaaaaggatgtcaacaaaccaatgcgtgtcttgcaagaagagggaattatcaaatttccggcaaacatcaatttcaaggattatttcgagtacagtatgttccccacgctagtctacacattgaacttccccagaattcgacatattagatgggcgtatgtgttgcagaaagttttgggaacatttgccttagtgtttgccatgattatcgtcgccgaagagagtttctgtcccttgatgcaagaagttgaacagtacacaagattgccaaccaaccaaaggttctcaaagtacttcgtcgttctttcccacttgatattgcccctcggcaaacagtacttgctctcgtttatcctcatttggaacgaaattctcaacgggatagcggagttaagcaggtttggggatcgccatttctacggcgcctggtggtcaagcgtcgactacatggactattcaagaaaatggaacacgatcgtgcaccgattcctccgccggcacgtttacaattcgaccattcgcatcctcggtatttccaggacccaagccgcgataattacacttttgctttcagccacaatccacgaactcgttatgtacatcctatttggaaaattacgagggtacctattccttacgatgcttgtccagatccccatgacagtcaccgccaagttcaacaaccgtttgtggggcaacatcatgttctggttgacgtatttatctggccccagcttggttagtgcgttgtatttactcttctga SEQ Acyl-CoAMSDDEIAGIVIEIDDDVKSTSSFQEDLVEVEMSNSSINESQTDELSYRPEEI ID SterolacylSLHSRRKSHKTPSDESFLEITKNVNDPDLVSKIENLRGKVSQREDRLRKHY NO:  transferaseLHTSQDVKFLSRFNDIKFKSNSATILDSDAFYKSEHFGVLTIFWVVIGLYIMS 46 (ARE2)TLSDMYFGMAKPLSDWIIIGMFKKDLMQVALVDLVMYLSSYFPYFLQVACK A.A. SeqTGAISWHGLGWAIQGVYSLVFLTFWAVLPSESAMDLPWIARVFLILHCLVFIMKMQSYGHYNGYLWDVYQEGLVSEADLTAVSEYDDDFPSDHGEVLEQSLWFAKHELEFQSNGTTERKDHHHHVFDEKDVNKPMRVLQEEGIIKFPANINFKDYFEYSMFPTLVYTLNFPRIRHIRWAYVLQKVLGTFALVFAMIIVAEESFCPLMQEVEQYTRLPTNQRFSKYFVVLSHLILPLGKQYLLSFILIWNEILNGIAELSRFGDRHFYGAWWSSVDYMDYSRKWNTIVHRFLRRHVYNSTIRILGISRTQAAIITLLLSATIHELVMYILFGKLRGYLFLTMLVQIPMTVTAKFNNRLWGNIMFWLTYLSGPSLVSALYLLF SEQ Diacylglycerolatgactcaggactataaagacgatagtcctacgtccactgagttggacactaacatagaagaggtgga IDacyltransferaseaagcactgcaaccctagagtcggaactcagacagagaaaacagaccacggaaactccagcatcaa NO: (DGA1) ccccaccaccacctccacaacaacagcaggcgcataagaaagccctgaagaatggcaagaggaa47 Nuc. Seqgagaccatttataaacgtggcgccgctcaacaccccgttggctcacaggctcgagactttggctgttgtttggcactgtgtcagtatcccgttctttatgtttttgttcttgcttacggtctccatggggttgcttgggtggttctttatcattttgccatatttcatttggtggtacggtttcgacttgcacactccatcgaatggtaaagttgtctatcgtgtgcgcaactcgttcaagaatttcatcatttgggactggtttgtcaagtatttcccgattgaagtgcacaagacggtcgagttggatcctacttttagcgaattgcctgtggaagagagcggcgacagttcggacgacgacgaacaagacttggtgtctgagcacagcagaactttggttgatcaaatcttcaagtttttcgggttgaagaaacgcttgaatgacacctccctgggcaaaccagagacattcaagaatgtgcctacgggtccaaggtatatttttgggtaccacccacacggagtgatttctatgggggcagtggggttgtttgccaacaacgccttgaggaacgaaccatatacgccaatttccaaatggttaaaaccattcttccacgacagctccaagggcgagagattgttccctggtattggcaatatcttcccattgacgcttaccacacagtttgcgctcccattttaccgtgactacttgatggctttggggatcactagtgcatcggctaaaaacattagaagcttgatcaacaatggagacaactctgtgtgtctcgtcgttggcggtgcacaagaatcgttgttgaacaatatgattgccaagcacgccagagtcgggtacggttacaaagagagcctagatattcatggcgaccagtccgaagaagaagaagaagaagaggatgataccaagcagctagagaacccaagtcctaaacgtgaagtgcaattggtcttgaacaaacgtaaaggttttgtgaagttggctatcgaactaggaaatgtttccttggtgcctatttttgcattcggagaagctgatgtttacagattggcccagccagcaccaggctcgttcttgtacaagttccagcaatggatgaaggcaacttttcaattcaccatcccattgtttagtgctcgaggcgtgttcatctatgatttcggattgttgccattcagaaacccaataaacatttgcgtcggtagacccgtctacattccgcacaacgtcttgcaagaatacaagcaaaagcacccagaggagtttgccgaagaggaacctgccagtaccccgatgaagaagtctggatctttcaccgatatgttcaaagctggtgaaaagaagcccaagacttcaagtatcaagactaaaatcccacctgcattactagacaagtaccacaagctatacgtcgacgagttgaagaaggtctatgaagagaacaaggaaaggtttggctacggtgatgttgaattaaacattgtagaatag SEQ DiacylglycerolMTQDYKDDSPTSTELDTNIEEVESTATLESELRQRKQTTETPASTPPPPP ID acyltransferaseQQQQAHKKASKNGKRKRPFINVAPLNTPLAHRLETLAVVWHCVSIPFFMF NO:  (DGA1)LFLLTVSMGLLGWFFIILPYFIWWYGFDLHTPSNGKVVYRVRNSFKNFIIWD 48 A.A. SeqWFVKYFPIEVHKTVELDPTFSELPVEESGDSSDDDEQDLVSEHSRTLVDQIFKFFGLKKRLNDTSSGKPETFKNVPTGPRYIFGYHPHGVISMGAVGLFANNALRNEPYTPISKWLKPFFHDSSKGERLFPGIGNIFPLTLTTQFALPFYRDYLMALGITSASAKNIRSLINNGDNSVCLVVGGAQESLLNNMIAKHARVGYGYKESLDIHGDQSEEEEEEEDDTKQLENPSPKREVQLVLNKRKGFVKLAIELGNVSLVPIFAFGEADVYRLAQPAPGSFLYKFQQWMKATFQFTIPLFSARGVFIYDFGLLPFRNPINICVGRPVYIPHNVLQEYKQKHPEEFAEEEPASTPMKKSGSFTDMFKAGEKKPKTSSIKTKIPPALLDKYHKLYVDELKKVYEENKERF GYGDVELNIVE SEQDiacylglycerolatgtcgtctttaaagaacagaaaatccgcaagcgtcgccacaagcgatacagaagactcagaaacag IDacyltransferaseaggcagtatcctcctcaattgatcccaacggcaccatattgcgaccagtcctacatgacgaaccccaccNO:  (LRO1)acagccatcaccaccacaacataactagaccagtattggaggacgatggcagcatcctggtgtccaga 49Nuc. Seqagatcgtcgatctccaaatccgacgacctgcaggcaaagcaaaagaagaagaaacccaagaagaagatcttggagtctcgtcgggtcatgtttatctttggtaccctcattgggttaatctttgcgtgggcgtttaccacagacacgcatcctttcaatggcgacttggagaagtttatcaactttgaccagctcaacgggatctttgacgactggaagaactggaaggatatcttgcccaacagcatccagacgtacttgcaggaatcgggcaagggcgaagataacgacgggttgcatggtctggccgattccttctccgtcgggctccgcttgaaagcccagaagaacttcactgacaaccacaatgtcgtgttggttcctggtgtggtgagcacggggttggaatcgtggggaacaaccaccaccggtgattgtccatctatcggatacttcaggaagagattgtggggatcattttatatgttaaggacaatgattttggagaaaacgtgctggttgaagcatatccagttggacgagaagacggggttggatcctcccaatattaaggtccgtgcggcgcagggtttcgaagcggcagatttctttatggctgggtactggatctggaacaagatcttgcagaacttggcggttattgggtacggaccaaataacatggtgagtgctagttatgactggagattggcttacattgacttggagagaagagatggatatttttcgaaacttaaagcgcagattgagttgaataacaagttgaacaacaagaagactgtgttgattggccactcgatggggacccagattattttctactttttgaaatgggtcgaagccaccgggaaaccatactatggcaatggcggaccaaactgggtgaatgatcatattgagtcgattattgacatcagtgggtcgactttgggtacccccaagagtattcctgtgttgatctctggggaaatgaaagacaccgttcaattgaacgcgttggcggtttacgggttggagcaatttttcagcaggcgtgaaagagtcgatatgttgcgtacatttggtggcgttgccagtatgttacccaaggggggagacaagatatggggcaacttgacgcatgcgccagatgatccaatttccacattcagtgatgacgaagttacggacagccacgaacctaaagatcgttcttttggtacgtttatccaattcaagaaccaaactagcgacgctaagccatacagggagatcaccatggctgaaggtatcgatgaattgttggacaaatcaccagactggtattccaagagagtccgtgagaactactcttacggcattacagacagcaaggcgcaattagagaagaacaacaatgaccacctgaagtggtcgaacccattagaagctgccttgcctaaagcacccgacatgaagatctattgtttctacggagttggaaatcctaccgaaagggcatacaagtatgtgactgccgataaaaaagccacgaaattggactacataatagacgccgacgatgccaatggagtcatattaggagacggagacggcactgtttcgttattaacccactcgatgtgccatgagtgggccaagggagacaagtcgagatacaacccagccaactcgaaggttaccattgttgaaatcaagcacgagccagacagatttgatttacgaggcggcgccaagactgcggaacatgttgatattttggggagtgccgagttgaacgagttgattttgactgtggttagcgggaacggggacgagattgagaatagatatgtcagcaacttaaaagaaatagtagaggccataaatttataa SEQDiacylglycerol MSSLKNRKSASVATSDTEDSETEAVSSSIDPNGTILRPVLHDEPHHSHHH IDacyltransferase HNITRPVLEDDGSISVSRRSSISKSDDSQAKQKKKKPKKKILESRRVMFIFGNO:  (LRO1) TLIGLIFAWAFTTDTHPFNGDLEKFINFDQLNGIFDDWKNWKDILPNSIQTY 50A.A. Seq LQESGKGEDNDGLHGSADSFSVGLRLKAQKNFTDNHNVVLVPGVVSTGLESWGTTTTGDCPSIGYFRKRLWGSFYMLRTMILEKTCWLKHIQLDEKTGLDPPNIKVRAAQGFEAADFFMAGYWIWNKILQNLAVIGYGPNNMVSASYDWRLAYIDLERRDGYFSKLKAQIELNNKLNNKKTVLIGHSMGTQIIFYFLKWVEATGKPYYGNGGPNWVNDHIESIIDISGSTLGTPKSIPVLISGEMKDTVQLNALAVYGLEQFFSRRERVDMLRTFGGVASMLPKGGDKIWGNLTHAPDDPISTFSDDEVTDSHEPKDRSFGTFIQFKNQTSDAKPYREITMAEGIDELLDKSPDWYSKRVRENYSYGITDSKAQLEKNNNDHSKWSNPLEAALPKAPDMKIYCFYGVGNPTERAYKYVTADKKATKLDYIIDADDANGVILGDGDGTVSLLTHSMCHEWAKGDKSRYNPANSKVTIVEIKHEPDRFDLRGGAKTAEHVDILGSAELNELILTVVSGNGDEIENRYVSNLKEIVEAINL SEQ ID ThioesteraseatggtggctgctgcagcaacttctgcattcttccccgttccagccccgggaacctcccctaaacccgNO: 51 activity,ggaagtccggcaactggccatcgagcttgagccctaccttcaagcccaagtcaatccccaatgct Cupheaggatttcaggttaaggcaaatgccagtgcccatcctaaggctaacggttctgcagtaaatctaaagtlanceolatactggcagcctcaacactcaggaggacacttcgtcgtcccctcctccccgggctttccttaaccagtt(Nucleic Acidgcctgattggagtatgcttctgactgcaatcacgaccgtcttcgtggcggcagagaagcagtggac Seq)tatgcttgataggaaatctaagaggcctgacatgctcgtggactcggttgggttgaagagtattgttcgggatgggctcgtgtccagacagagttttttgattagatcttatgaaataggcgctgatcgaacagcctctatagagacgctgatgaaccacttgcaggaaacatctatcaatcattgtaagagtttgggtcttctcaatgacggctttggtcgtactcctgggatgtgtaaaaacgacctcatttgggtgcttacaaaaatgcagatcatggtgaatcgctacccaacttggggcgatactgttgagatcaatacctggttctctcagtcggggaaaatcggtatggctagcgattggctaataagtgattgcaacacaggagaaattcttataagagcaacgagcgtgtgggctatgatgaatcaaaagacgagaagattctcaagacttccatacgaggttcgccaggagttaacacctcattttgtggactctcctcatgtcattgaagacaatgatcagaaattgcataagtttgatgtgaagactggtgattccattcgcaagggtctaactccgaggtggaatgacttggatgtgaatcagcacgtaagcaacgtgaagtacattgggtggattctcgagagtatgccaatagaagttttggagacccaggagctatgctctctcaccgttgaatataggcgggaatgcggaatggacagtgtgctggagtccgtgactgctgtggatccctcagaaaatggaggccggtctcagtacaagcaccttttgcggcttgaggatgggactgatatcgtgaagagcagaactgagtggcgaccgaagaatgcaggaactaacggggcgatatcaacatcaacagcaaagacttcaaatggaaactcggcctcttag SEQ IDCYP52Al2, MATQEIIDSVLPYLTKWYTVITAAVLVFLISTNIKNYVKAKKLKCandidaD NO: 52ATCC20336 PPYLKDAGLTGISSLIAAIKAKNDGRLANFADEVFDEYPNHTFYLSVAG (Amino AcidALKIVMTVDPENIKAVLATQFTDFSLGTRHAHFAPLLGDGIFTLDGEG Seq.)WKHSRAMLRPQFARDQIGHVKALEPHIQIMAKQIKLNQGKTFDIQELFFRFTVDTATEFLFGESVHSLYDEKLGIPTPNEIPGRENFAAAFNVSQHYLATRSYSQTFYFLTNPKEFRDCNAKVHHLAKYFVNKALNFTPEELEEKSKSGYVFLYELVKQTRDPKVLQDQLLNIMVAGRDTTAGLLSFALFELARHPEMWSKLREEIEVNFGVGEDSRVEEITFEALKRCEYLKAILNETLRMYPSVPVNFRTATRDTTLPRGGGANGTDPIYIPKGSTVAYVVYKTHRLEEYYGKDANDFRPERWFEPSTKKLGWAYVPFNGGPRVCLGQQFALTEASYVITRLAQMFETVSSDPGLEYPPPKCIHLTMSHNDGVFVKM* SEQ ID CYP52A13,MTVHDIIATYFTKWYVIVPLALIAYRVLDYFYGRYLMYKLGAKPFFQKQ NO: 53 ATCC20336TDGCFGFKAPLELLKKKSDGTLIDFTLQRIHDLDRPDIPTFTFPVFSINL (Amino AcidVNTLEPENIKAILATQFNDFSLGTRHSHFAPLLGDGIFTLDGAGWKHS Seq.)RSMLRPQFAREQISHVKLLEPHVQVFFKHVRKAQGKTFDIQELFFRLTVDSATEFLFGESVESLRDESIGMSINALDFDGKAGFADAFNYSQNYLASRAVMQQLYWVLNGKKFKECNAKVHKFADYYVNKALDLTPEQLEKQDGYVFLYELVKQTRDKQVLRDQLLNIMVAGRDTTAGLLSFVFFELARNPEVTNKLREEIEDKFGLGENASVEDISFESLKSCEYLKAVLNETLRLYPSVPQNFRVATKNTTLPRGGGKDGLSPVLVRKGQTVIYGVYAAHRNPAVYGKDALEFRPERWFEPETKKLGWAFLPFNGGPRICLGQQFALTEASYVTVRLLQEFAHLSMDPDTEYPPKKMSHLTMSLFDGANIEMY* SEQ ID CYP52A14,MTAQDIIATYITKWYVIVPLALIAYRVLDYFYGRYLMYKLGAKPFFQKQ NO: 54 ATCC20336TDGYFGFKAPLELLKKKSDGTLIDFTLERIQALNRPDIPTFTFPIFSINLI (Amino AcidSTLEPENIKAILATQFNDFSLGTRHSHFAPLLGDGIFTLDGAGWKHSR Seq.)SMLRPQFAREQISHVKLLEPHMQVFFKHVRKAQGKTFDIQELFFRLTVDSATEFLFGESVESLRDESIGMSINALDFDGKAGFADAFNYSQNYLASRAVMQQLYWVLNGKKFKECNAKVHKFADYYVSKALDLTPEQLEKQDGYVFLYELVKQTRDRQVLRDQLLNIMVAGRDTTAGLLSFVFFELARNPEVTNKLREEIEDKFGLGENARVEDISFESLKSCEYLKAVLNETLRLYPSVPQNFRVATKNTTLPRGGGKDGLSPVLVRKGQTVMYGVYAAHRNPAVYGKDALEFRPERWFEPETKKLGWAFLPFNGGPRICLGQQFALTEASYVTVRLLQEFGHLSMDPNTEYPPRKMSHLTMSLFDGANIEMY* SEQ ID CYP52A15,MSSSPSFAQEVLATTSPYIEYFLDNYTRWYYFIPLVLLSLNFISLLHTRY NO: 55 ATCC20336LERRFHAKPLGNFVRDPTFGIATPLLLIYLKSKGTVMKFAWGLWNNKY (Amino AcidIVRDPKYKTTGLRIVGLPLIETMDPENIKAVLATQFNDFSLGTRHDFLY Seq.)SLLGDGIFTLDGAGWKHSRTMLRPQFAREQVSHVKLLEPHVQVFFKHVRKHRGQTFDIQELFFRLTVDSATEFLFGESAESLRDESIGLTPTTKDFDGRRDFADAFNYSQTYQAYRFLLQQMYWILNGSEFRKSIAVVHKFADHYVQKALELTDDDLQKQDGYVFLYELAKQTRDPKVLRDQLLNILVAGRDTTAGLLSFVFYELSRNPEVFAKLREEVENRFGLGEEARVEEISFESLKSCEYLKAVINETLRLYPSVPHNFRVATRNTTLPRGGGEDGYSPIVVKKGQVVMYTVIATHRDPSIYGADADVFRPERWFEPETRKLGWAYVPFNGGPRICLGQQFALTEASYVTVRLLQEFAHLSMDPDTEYPPKLQNTL TLSLFDGADVRMY* SEQ IDCYP52A16, MSSSPSFAQEVLATTSPYIEYFLDNYTRWYYFIPLVLLSLNFISLLHTKY NO: 56ATCC20336 LERRFHAKPLGNVVLDPTFGIATPLILIYLKSKGTVMKFAWSFWNNKYI (Amino AcidVKDPKYKTTGLRIVGLPLIETIDPENIKAVLATQFNDFSLGTRHDFLYSL Seq.)LGDGIFTLDGAGWKHSRTMLRPQFAREQVSHVKLLEPHVQVFFKHVRKHRGQTFDIQELFFRLTVDSATEFLFGESAESLRDDSVGLTPTTKDFEGRGDFADAFNYSQTYQAYRFLLQQMYWILNGAEFRKSIAIVHKFADHYVQKALELTDDDLQKQDGYVFLYELAKQTRDPKVLRDQLLNILVAGRDTTAGLLSFVFYELSRNPEVFAKLREEVENRFGLGEEARVEEISFESLKSCEYLKAVINEALRLYPSVPHNFRVATRNTTLPRGGGKDGCSPIVVKKGQVVMYTVIGTHRDPSIYGADADVFRPERWFEPETRKLGWAYVPFNGGPRICLGQQFALTEASYVTVRLLQEFGNLSSDPNAEYPPKLQNT LTLSLFDGADVRMF* SEQ IDCYP52A17, MIEQLLEYWYVVVPVLYIIKQLLAYTKTRVLMKKLGAAPVTNKLYDNAF NO: 57ATCC20336 GIVNGWKALQFKKEGRAQEYNDYKFDHSKNPSVGTYVSILFGTRIVV (Amino AcidTKDPENIKAILATQFGDFSLGKRHTLFKPLLGDGIFTLDGEGWKHSRA Seq.)MLRPQFAREQVAHVTSLEPHFQLLKKHILKHKGEYFDIQELFFRFTVDSATEFLFGESVHSLKDESIGINQDDIDFAGRKDFAESFNKAQEYLAIRTLVQTFYWLVNNKEFRDCTKSVHKFTNYYVQKALDASPEELEKQSGYVFLYELVKQTRDPNVLRDQSLNILLAGRDTTAGLLSFAVFELARHPEIWAKLREEIEQQFGLGEDSRVEEITFESLKRCEYLKAFLNETLRIYPSVPRNFRIATKNTTLPRGGGSDGTSPILIQKGEAVSYGINSTHLDPVYYGPDAAEFRPERWFEPSTKKLGWAYLPFNGGPRICLGQQFALTEAGYVLVRLVQEFSHVRSDPDEVYPPKRLTNLTMCLQDGAIVKFD* SEQ ID CYP52A18,MIEQILEYWYIVVPVLYIIKQLIAYSKTRVLMKQLGAAPITNQLYDNVFGI NO: 58 ATCC20336VNGWKALQFKKEGRAQEYNDHKFDSSKNPSVGTYVSILFGTKIVVTK (Amino AcidDPENIKAILATQFGDFSLGKRHALFKPLLGDGIFTLDGEGWKHSRSML Seq.)RPQFAREQVAHVTSLEPHFQLLKKHILKHKGEYFDIQELFFRFTVDSATEFLFGESVHSLKDETIGINQDDIDFAGRKDFAESFNKAQEYLSIRILVQTFYWLINNKEFRDCTKSVHKFTNYYVQKALDATPEELEKQGGYVFLYELVKQTRDPKVLRDQSLNILLAGRDTTAGLLSFAVFELARNPHIWAKLREEIEQQFGLGEDSRVEEITFESLKRCEYLKAFLNETLRVYPSVPRNFRIATKNTTLPRGGGPDGTQPILIQKGEGVSYGINSTHLDPVYYGPDAAEFRPERWFEPSTRKLGWAYLPFNGGPRICLGQQFALTEAGYVLVRLVQEFSHIRSDPDEVYPPKRLTNLTMCLQDGAIVKFD* SEQ ID CYP52A19,MLDQILHYWYIVLPLLAIINQIVAHVRTNYLMKKLGAKPFTHVQRDGWL NO: 59 ATCC20336GFKFGREFLKAKSAGRSVDLIISRFHDNEDTFSSYAFGNHVVFTRDPE (Amino AcidNIKALLATQFGDFSLGSRVKFFKPLLGYGIFTLDAEGWKHSRAMLRPQ Seq.)FAREQVAHVTSLEPHFQLLKKHILKHKGEYFDIQELFFRFTVDSATEFLFGESVHSLKDEEIGYDTKDMSEERRRFADAFNKSQVYVATRVALQNLYWLVNNKEFKECNDIVHKFTNYYVQKALDATPEELEKQGGYVFLYELVKQTRDPKVLRDQSLNILLAGRDTTAGLLSFAVFELARNPHIWAKLREEIEQQFGLGEDSRVEEITFESLKRCEYLKAVLNETLRLHPSVPRNARFAIKDTTLPRGGGPNGKDPILIRKDEVVQYSISATQTNPAYYGADAADFRPERWFEPSTRNLGWAFLPFNGGPRICLGQQFALTEAGYVLVRLVQEFPNLSQDPETKYPPPRLAHLTMCLFDGAHVKMS* SEQ ID CYP52A20,MLDQIFHYWYIVLPLLVIIKQIVAHARTNYLMKKLGAKPFTHVQLDGWF NO: 60 ATCC20336GFKFGREFLKAKSAGRQVDLIISRFHDNEDTFSSYAFGNHVVFTRDPE (Amino AcidNIKALLATQFGDFSLGSRVKFFKPLLGYGIFTLDGEGWKHSRAMLRP Seq.)QFAREQVAHVTSLEPHFQLLKKHILKHKGEYFDIQELFFRFTVDSATEFLFGESVHSLRDEEIGYDTKDMAEERRKFADAFNKSQVYLSTRVALQTLYWLVNNKEFKECNDIVHKFTNYYVQKALDATPEELEKQGGYVFLYELAKQTKDPNVLRDQSLNILLAGRDTTAGLLSFAVFELARNPHIWAKLREEIESHFGSGEDSRVEEITFESLKRCEYLKAVLNETLRLHPSVPRNARFAIKDTTLPRGGGPNGKDPILIRKNEVVQYSISATQTNPAYYGADAADFRPERWFEPSTRNLGWAYLPFNGGPRICLGQQFALTEAGYVLVRLVQEFPSLSQDPETEYPPPRLAHLTMCLFDGAYVKMQ* SEQ ID CYP52D2,MAISSLLSWDVICandidaVFICandidaCandidaYFGYEYCYTKYLMHKH NO: 61 ATCC20336GAREIENVINDGFFGFRLPLLLMRASNEGRLIEFSVKRFESAPHPQNK (Amino AcidTLVNRALSVPVILTKDPVNIKAMLSTQFDDFSLGLRLHQFAPLLGKGIF Seq.)TLDGPEWKQSRSMLRPQFAKDRVSHISDLEPHFVLLRKHIDGHNGDYFDIQELYFRFSMDVATGFLFGESVGSLKDEDARFSEAFNESQKYLATRATLHELYFLCDGFRFRQYNKVVRKFCSQCandidaHKALDVAPEDTSEYVFLRELVKHTRDPVVLQDQALNVLLAGRDTTASLLSFATFELARNDHMWRKLREEVISTMGPSSDEITVAGLKSCRYLKAILNETLRLYPSVPRNARFATRNTTLPRGGGPDGSFPILIRKGQPVGYFICATHLNEKVYGNDSHVFRPERWAALEGKSLGWSYLPFNGGPRSCLGQQFAILEASYVLARLTQCYTTIQLRTTEYPPKKLVHLTMSLLNGVYIRTRT* SEQ ID ADH1-1,MHALFSKSVFLKYVSSPTTSAIPHSSEFIVPRSFYLRRSISPYLPHSSLF NO: 62 CandidaPSFSYSSSSVYTKKSFHTMSANIPKTQKAVVFEKNGGELEYKDIPVPT (Amino AcidPKANELLINVKYSGVCHTDLHAWKGDWPLATKLPLVGGHEGAGVVV Seq.)GMGENVKGWKIGDFAGIKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGSFEQYATADAVQAARIPAGTDLAEVAPILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGAFVKSLGAEAYIDFLKEKDIVSAVKKATDGGPHGAINVSVSEKAIDQSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGLSELPQVFKLMEEGKILGRYVLDTSK SEQ ID NADPHMALDKLDLYVIITLVVAIAAYFAKNQFLDQQQDTGFLNTDSGDGNSRDI NO: 63 cytochromeLQALKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFGLKTMVADFA P450DYDFENFGDITEDILVFFIVATYGEGEPTDNADEFHTWLTEEADTLSTL reductase,KYTVFGLGNSTYEFFNAIGRKFDRLLGEKGGDRFAEYGEGDDGTGTL CPR (CandidaDEDFLAWKDNVFDSLKNDLNFEEKELKYEPNVKLTERDDLSGNDPDV strainSLGEPNVKYIKSEGVDLTKGPFDHTHPFLARIVKTKELFTSEDRHCand ATCC750)idaHVEFDISESNLKYTTGDHLAIWPSNSDENIKQFAKCFGLEDKLDTVI (Amino AcidELKALDSTYSIPFPNPITYGAVIRHHLEISGPVSRQFFLSIAGFAPDEET Seq.)KKSFTRIGGDKQEFASKVTRRKFNIADALLFASNNRPWSDVPFEFLIENVQHLTPRYYSISSSSLSEKQTINVTAVVEAEEEADGRPVTGVVTNLLKNIEIEQNKTGETPMVHYDLNGPRGKFSKFRLPVHVRRSNFKLPKNSTTPVILIGPGTGVAPLRGFVRERVQQVKNGVNVGKTVLFYGCRNSEQDFLYKQEWSEYASVLGENFEMFNAFSRQDPTKKVYVQDKILENSALVDELLSSGAIIYVCGDASRMARDVQAAIAKIVAKSRDIHEDKAAELVKSW KVQNRYQEDVW SEQ IDNADPH MALDKLDLYVIITLVVAVAAYFAKNQFLDQPQDTGFLNTDSGSNSRDV NO: 64 cytochromeLSTLKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFGLKTMVADFA P450DYDWDNFGDITEDILVFFIVATYGEGEPTDNADEFHTWLTEEADTLST reductase A,LKYTVFGLGNSTYEFFNAIGRKFDRLLSEKGGDRFAEYAEGDDGTGT CPRALDEDFMAWKDNVFDALKNDLNFEEKELKYEPNVKLTERDDLSAADSQ (CandidaVSLGEPNKKYINSEGIDLTKGPFDHTHPYLARITETRELFSSKDRHCIH strainVEFDISESNLKYTTGDHLAIWPSNSDENIKQFAKCFGLEDKLDTVIELK ATCC20336)ALDSTYTIPFPTPITYGAVIRHHLEISGPVSRQFFLSIAGFAPDEETKKA (Amino AcidFTRLGGDKQEFAAKVTRRKFNIADALLYSSNNAPWSDVPFEFLIENVP Seq.)HLTPRYYSISSSSLSEKQLINVTAVVEAEEEADGRPVTGVVTNLLKNVEIVQNKTGEKPLVHYDLSGPRGKFNKFKLPVHVRRSNFKLPKNSTTPVILIGPGTGVAPLRGFVRERVQQVKNGVNVGKTLLFYGCRNSNEDFLYKQEWAEYASVLGENFEMFNAFSRQDPSKKVYVQDKILENSQLVHELLTEGAIIYVCGDASRMARDVQTTISKIVAKSREISEDKAAELVKSWKVQ NRYQEDVW SEQ ID NADPHMALDKLDLYVIITLVVAVAAYFAKNQFLDQPQDTGFLNTDSGSNSRDV NO: 65 cytochromeLSTLKKNNKNTLLLFGSQTGTAEDYANKLSRELHSRFGLKTMVADFA P450DYDWDNFGDITEDILVFFIVATYGEGEPTDNADEFHTWLTEEADTLST reductase B,LRYTVFGLGNSTYEFFNAIGRKFDRLLSEKGGDRFAEYAEGDDGTGT CPRBLDEDFMAWKDNVFDALKNDLNFEEKELKYEPNVKLTERDDLSAADSQ (CandidaVSLGEPNKKYINSEGIDLTKGPFDHTHPYLARITETRELFSSKERHCIH strainVEFDISESNLKYTTGDHLAIWPSNSDENIKQFAKCFGLEDKLDTVIELK ATCC20336)ALDSTYTIPFPTPITYGAVIRHHLEISGPVSRQFFLSIAGFAPDEETKKT (Amino AcidFTRLGGDKQEFATKVTRRKFNIADALLYSSNNTPWSDVPFEFLIENIQ Seq.)HLTPRYYSISSSSLSEKQLINVTAVVEAEEEADGRPVTGVVTNLLKNIEIAQNKTGEKPLVHYDLSGPRGKFNKFKLPVHVRRSNFKLPKNSTTPVILIGPGTGVAPLRGFVRERVQQVKNGVNVGKTLLFYGCRNSNEDFLYKQEWAEYASVLGENFEMFNAFSRQDPSKKVYVQDKILENSQLVHELLTEGAIIYVCGDASRMARDVQTTISKIVAKSREISEDKAAELVKSWKVQN RYQEDVW SEQ IDNADPHP450atgacaattaaagaaatgcctcagccaaaaacgtttggagagcttaaaaatttaccgttattaaacNO: 66 cytochromeacagataaaccggttcaagctttgatgaaaattgcggatgaattaggagaaatctttaaattcgagreductasegcgcctggtcgtgtaacgcgctacttatcaagtcagcgtctaattaaagaagcatgcgatgaatca(Bacilluscgctttgataaaaacttaagtcaagcgcttaaatttgtacgtgattttgcaggagacgggttatttacamegaterium)agctggacgcatgaaaaaaattggaaaaaagcgcataatatcttacttccaagcttcagtcagca(Nucleid Acidggcaatgaaaggctatcatgcgatgatggtcgatatcgccgtgcagcttgttcaaaagtgggagc Seq.)gtctaaatgcagatgagcatattgaagtaccggaagacatgacacgtttaacgcttgatacaattggtctttgcggctttaactatcgctttaacagcttttaccgagatcagcctcatccatttattacaagtatggtccgtgcactggatgaagcaatgaacaagctgcagcgagcaaatccagacgacccagcttatgatgaaaacaagcgccagtttcaagaagatatcaaggtgatgaacgacctagtagataaaattattgcagatcgcaaagcaagcggtgaacaaagcgatgatttattaacgcatatgctaaacggaaaagatccagaaacgggtgagccgcttgatgacgagaacattcgctatcaaattattacattcttaattgcgggacacgaaacaacaagtggtcttttatcatttgcgctgtatttcttagtgaaaaatccacatgtattacaaaaagcagcagaagaagcagcacgagttctagtagatcctgttccaagctacaaacaagtcaaacagcttaaatatgtcggcatggtcttaaacgaagcgctgcgcttatggccaactgctcctgcgttttccctatatgcaaaagaagatacggtgcttggaggagaatatcctttagaaaaaggcgacgaactaatggttctgattcctcagcttcaccgtgataaaacaatttggggagacgatgtggaagagttccgtccagagcgttttgaaaatccaagtgcgattccgcagcatgcgtttaaaccgtttggaaacggtcagcgtgcgtgtatcggtcagcagttcgctcttcatgaagcaacgctggtacttggtatgatgctaaaacactttgactttgaagatcatacaaactacgagctggatattaaagaaactttaacgttaaaacctgaaggctttgtggtaaaagcaaaatcgaaaaaaattccgcttggcggtattccttcacctagcactgaacagtctgctaaaaaagtacgcaaaaaggcagaaaacgctcataatacgccgctgcttgtgctatacggttcaaatatgggaacagctgaaggaacggcgcgtgatttagcagatattgcaatgagcaaaggatttgcaccgcaggtcgcaacgcttgattcacacgccggaaatcttccgcgcgaaggagctgtattaattgtaacggcgtcttataacggtcatccgcctgataacgcaaagcaatttgtcgactggttagaccaagcgtctgctgatgaagtaaaaggcgttcgctactccgtatttggatgcggcgataaaaactgggctactacgtatcaaaaagtgcctgcttttatcgatgaaacgcttgccgctaaaggggcagaaaacatcgctgaccgcggtgaagcagatgcaagcgacgactttgaaggcacatatgaagaatggcgtgaacatatgtggagtgacgtagcagcctactttaacctcgacattgaaaacagtgaagataataaatctactctttcacttcaatttgtcgacagcgccgcggatatgccgcttgcgaaaatgcacggtgcgttttcaacgaacgtcgtagcaagcaaagaacttcaacagccaggcagtgcacgaagcacgcgacatcttgaaattgaacttccaaaagaagcttcttatcaagaaggagatcatttaggtgttattcctcgcaactatgaaggaatagtaaaccgtgtaacagcaaggttcggcctagatgcatcacagcaaatccgtctggaagcagaagaagaaaaattagctcatttgccactcgctaaaacagtatccgtagaagagcttctgcaatacgtggagcttcaagatcctgttacgcgcacgcagcttcgcgcaatggctgctaaaacggtctgcccgccgcataaagtagagcttgaagccttgcttgaaaagcaagcctacaaagaacaagtgctggcaaaacgtttaacaatgcttgaactgcttgaaaaatacccggcgtgtgaaatgaaattcagcgaatttatcgcccttctgccaagcatacgcccgcgctattactcgatttcttcatcacctcgtgtcgatgaaaaacaagcaagcatcacggtcagcgttgtctcaggagaagcgtggagcggatatggagaatataaaggaattgcgtcgaactatcttgccgagctgcaagaaggagatacgattacgtgctttatttccacaccgcagtcagaatttacgctgccaaaagaccctgaaacgccgcttatcatggtcggaccgggaacaggcgtcgcgccgtttagaggctttgtgcaggcgcgcaaacagctaaaagaacaaggacagtcacttggagaagcacatttatacttcggctgccgttcacctcatgaagactatctgtatcaagaagagcttgaaaacgcccaaagcgaaggcatcattacgcttcataccgctttttctcgcatgccaaatcagccgaaaacatacgttcagcacgtaatggaacaagacggcaagaaattgattgaacttcttgatcaaggagcgcacttctatatttgcggagacggaagccaaatggcacctgccgttgaagcaacgcttatgaaaagctatgctgacgttcaccaagtgagtgaagcagacgctcgcttatggctgcagcagctagaagaaaaaggccgatacgcaaaagacgtgtgggctggg SEQ ID IDP2,MGEIQKITVKNPIVEMDGDEMTRIIWQFIKDKLILPYLNVDLKYYDLGIE NO: 67 CandidaYRDQTDDKVTTDAAEAILKYGVGVKCATITPDEARVKEFNLKKMWLS (Amino AcidPNGTLRNVLGGTVFREPIVIDNIPRIVPSWEKPIIIGRHAFGDQYKATDV Seq.)VIPAAGDLKLVFKPKDGGEVQEYPVYQFDGRGVALSMYNTDASITDFAESSFQLAIERKLNLFSSTKNTILKKYDGKFKDIFEGLYASKYKTKMDELGIWYEHRLIDDMVAQMLKSKGGYIIAMKNYDGDVQSDIVAQGFGSLGLMTSVLVTPDGKAFESEAAHGTVTRHYRQHQQGKETSTNSIASIYAWTRGLVQRGKLDDTPEVVKFAEELEKAVIETVSKDNIMTKDLALTQGKTDRSSYVTTEEFIDGVANRLNKNLGY SEQ ID IDP2,atgggcgaaattcagaaaataacagtcaagaacccaatcgtcgaaatggacggtgacgaaatg NO: 68Candidaacccgtatcatctggcaattcatcaaagacaagttgatcttgccttatttgaacgttgacttgaaatact(Nucleic Acidacgacttgggcatcgagtacagagaccagaccgacgacaaggtcaccaccgatgctgctgaa Seq.)gccatcttgaaatacggtgtcggtgtcaagtgtgccaccatcaccccagacgaagccagagttaaagaattcaacttgaagaagatgtggctctctccaaacggtactttgagaaacgtccttggtggtactgtcttcagagaaccaattgtcattgacaacatcccaagaatcgtgcctagctgggaaaaaccaatcatcattggtagacacgctttcggcgaccaatacaaggccaccgacgtggttatcccagctgccggtgacttgaagttggtgttcaagccaaaagacggcggtgaagtgcaagaatacccagtctaccagttcgacggtcgaggtgtcgccttgagcatgtacaacaccgacgcttcaatcactgatttcgctgaaagttccttccaattggccattgagcgtaaattgaacttgttttcttccaccaaaaacaccatcttgaagaaatacgacgggaaattcaaagacatattcgaaggcttgtacgccagcaaatacaagaccaagatggacgaattgggcatctggtacgagcacagattgattgacgatatggttgcacagatgttgaagtctaaaggtggttacatcatcgccatgaaaaactacgatggtgatgtccaatccgacattgtcgcacaaggtttcggttccttgggtttgatgacctctgttttggttaccccagacggcaaggcttttgaatccgaggctgcccacggtactgtcactagacattatagacaacaccaacaaggtaaagagacctcgaccaactccattgcctccatctacgcctggaccagagggttggtccaaagaggtaagttggatgacactccggaagttgtcaagtttgctgaagagttggaaaaggctgtcattgagactgtctccaaggacaacatcatgaccaaagatttggccttgacccaaggtaagaccgacagatcttcgtatgtcacgactgaagaattcatcgatggtgttgctaatagattgaacaaaaacttgggctac SEQ ID IDP3, ScMSKIKVVHPIVEMDGDEQTRVIWKLIKEKLILPYLDVDLKYYDLSIQERD NO: 69 (Amino AcidRTNDQVTKDSSYATLKYGVAVKCATITPDEARMKEFNLKEMWKSPN Seq.)GTIRNILGGTVFREPIIIPKIPRLVPHWEKPIIIGRHAFGDQYRATDIKIKKAGKLRLQFSSDDGKENIDLKVYEFPKSGGIAMAMFNTNDSIKGFAKASFELALKRKLPLFFTTKNTILKNYDNQFKQIFDNLFDKEYKEKFQALKITYEHRLIDDMVAQMLKSKGGFIIAMKNYDGDVQSDIVAQGFGSLGLMTSILITPDGKTFESEAAHGTVTRHFRKHQRGEETSTNSIASIFAWTRAIIQRGKLDNTDDVIKFGNLLEKATLDTVQVGGKMTKDLALMLGKTNRSSYVTTEEFIDEVAKRLQNMMLSSNEDKKGMCKL* SEQ ID IDP3, ScatgagtaaaattaaagttgttcatcccatcgtggaaatggacggtgatgagcagacaagagttatttNO: 70 (Nucleic AcidggaaacttatcaaagaaaaattgatattgccatatttagatgtggatttaaaatactatgacctttcaaSeq.)tccaagagcgtgataggactaatgatcaagtaacaaaggattcttcttatgctaccctaaaatatggggttgctgtcaaatgtgccactataacacccgatgaggcaagaatgaaagaatttaaccttaaagaaatgtggaaatctccaaatggaacaatcagaaacatcctaggtggaactgtatttagagaacccatcattattccaaaaatacctcgtctagtccctcactgggagaaacctataattataggccgtcatgcttttggtgaccaatatagggctactgacatcaagattaaaaaagcaggcaaactaaggttacagtttagctcagatgacggtaaagaaaacatcgatttaaaggtttatgaatttcctaaaagtggtgggatcgcaatggcaatgtttaatacaaatgattccattaaagggttcgcaaaggcatccttcgaattagctctcaaaagaaaactaccgttattctttacaaccaaaaacactattctgaaaaattatgataatcagttcaaacaaattttcgataatttgttcgataaagaatataaggaaaagtttcaggctttaaaaataacgtacgagcatcgtttgattgatgatatggtagcacagatgctaaaatcaaagggcgggtttataatcgccatgaagaattatgatggcgatgtccagtctgacattgtggcacaaggatttgggtctcttggtttaatgacgtccatattgattacacctgatggtaaaacgtttgaaagcgaggctgcccatggtacggtgaccagacattttagaaaacatcaaagaggcgaagaaacatcaacaaattcaatagcctcaatatttgcctggacaagggcaattatacaaagaggaaaattagacaatacagatgatgttataaaatttggaaacttactagaaaaggctactttggacacagttcaagtgggcggaaaaatgaccaaggatttagcattgatgcttggaaagactaatagatcatcatatgtaaccacagaagagtttattgatgaagttgccaagaggcttcaaaacatgatgctcagctccaatgaagacaagaaaggtatgtgcaaactataa SEQ IDGDP1, KIatgcccgatatgacaaacgaatcttcttctaagccagctcaaattaacattggtatcaatggttttggtNO: 71 (Nucleic AcidagaatcggtagattggttctacgtgctgctttgacgcacccagaagttaaggtcagattaatcaataSeq.)atccatccacaacaccagaatacgctgcttatttgttcaaatacgattctactcacggcaagtatcgtggtgaagttgaattcgacgatgaacgtatcatcattcaaaatgaccatgtttcggctcatatccctctatctcattttagggaaccagagcgtatcccatgggcttcctacaacgtcgattatgtaattgactcaaccggtgtcttcaaggaagtcgatacagcctctagacataaaggtgtcaaaaaagttatcattactgctccatcaaagaccgcgccaatgtacgtctatggtgttaaccacgttaaatacaacccattgacggatcacgtggtctctaatgcctcctgtactaccaactgtttggctccgttggttaaggctttggacgatgagttcggtatcgaagaagccttgatgacaactattcatgcaactactgcttctcaaaagactgtcgatggtaccagttctggtggtaaggactggagaggcggtagatcttgccagggaaatatcattccttcatctactggtgcagctaaggctgtagggaaaatcttgcctgaacttaatggtaagatcaccggtatgtctataagagtcccaacaattaatatttccctggttgacttgacattccgtacagcaaagaaaacttcttacgatgacattatgaaggccctagaacaaagatctcgcagcgatatgaagggtgttttgggtgttaccaaagacgccgttgtgtcctctgacttcacatccgattcacgttcatctattgttgatgccaaggccggtattgaattgaacgaccattttttcaaggtcctttcttggtatgataatgaatatggttactcttcaagagtggttgatttatccattttcatggctcaaaaggacttcgaagctggtgtttaa SEQ ID GDP1, KIMPDMTNESSSKPAQINIGINGFGRIGRLVLRAALTHPEVKVRLINNPST NO: 72 (Amino AcidTPEYAAYLFKYDSTHGKYRGEVEFDDERIIIQNDHVSAHIPLSHFREPE Seq.)RIPWASYNVDYVIDSTGVFKEVDTASRHKGVKKVIITAPSKTAPMYVYGVNHVKYNPLTDHVVSNASCTTNCLAPLVKALDDEFGIEEALMTTIHATTASQKTVDGTSSGGKDWRGGRSCQGNIIPSSTGAAKAVGKILPELNGKITGMSIRVPTINISLVDLTFRTAKKTSYDDIMKALEQRSRSDMKGVLGVTKDAVVSSDFTSDSRSSIVDAKAGIELNDHFFKVLSWYDNEYGYS SRVVDLSIFMAQKDFEAGVSEQ ID ZWF1,atgtcttatgattcattcggtgactacgtcactatcgtcgttttcggtgcttccggtgacttggccagcaaNO: 73 Candidaaaaaaccttccctgccttgtttggcttgtttagagaaaagcaattgcccccaaccgtccagatcattg(Nucleic Acidgctatgccagatcccatttgtccgacaaggacttcaaaaccaagatctcctcccacttcaagggcg Seq.)gcgacgaaaaaaccaagcaagacttcttgaacttgtgtacttatatcagcgacccatacgacactgacgatggttacaagagattggaagccgccgctcaagaatacgaatccaagcacaacgtcaaggtccctgaaagattgttttacttggccttgcctccttctgtcttccacaccgtctgtgagcaagtcaagaagatcgtctaccctaaggacggtaagctcagaatcatcattgaaaagccgttcggacgtgatttggccacctaccgtgaattgcaaaagcaaatctccccattgttcaccgaagacgaactctacagaattgaccactacttgggtaaagaaatggtcaagaacttgttggttttgagattcggtaacgaattgttcagtgggatctggaacaacaagcacatcacctcggtgcaaatctccttcaaggaacccttcggtaccgaaggtagaggtggctactttgacaacattggtatcatcagagatgtcatgcaaaaccacttgttgcaagtcttgaccttgttgaccatggaaagaccagtctcttttgacccagaagctgtcagagacgaaaaggtcaaggttttgaaagcttttgacaagattgacgtcaacgacgttcttttgggacaatacgccaagtctgaggatggctccaagccaggttacttggatgactccaccgtcaagccaaactccaaggctgtcacctacgccgctttcagagtcaacatccacaacgaaagatgggacggtgttccaattgttttgagagccggtaaggctttagacgaaggtaaagttgaaattagaatccaattcaagccagttgccaaaggtatgtttaaggagatccaaagaaacgaattggttattagaatccaaccagacgaagccatctacttgaagatcaactccaagatcccaggtatctccaccgaaacttccttgaccgacttggacttgacttactccaagcgttactccaaggacttctggatcccagaagcatacgaagccttgatcagagactgttacttgggcaaccactccaactttgtcagagacgatgaattggaagttgcttggaagctcttcaccccattgttggaagccgttgaaaaagaagacgaagtcagcttgggaacctacccatacggatccaagggtcctaaagaattgagaaagtacttggtcgaccacggttacgtcttcaacgacccaggtacttaccaatggccattgaccaacaccgatgtcaaaggtaagatctaa SEQ ID ZWF1,MSYDSFGDYVTIVVFGASGDLASKKTFPALFGLFREKQLPPTVQIIGY NO: 74 CandidaARSHLSDKDFKTKISSHFKGGDEKTKQDFLNLCTYISDPYDTDDGYKR (Amino AcidLEAAAQEYESKHNVKVPERLFYLALPPSVFHTVCEQVKKIVYPKDGKL Seq.)RIIIEKPFGRDLATYRELQKQISPLFTEDELYRIDHYLGKEMVKNLLVLRFGNELFSGIWNNKHITSVOISFKEPFGTEGRGGYFDNIGIIRDVMQNHLLQVLTLLTMERPVSFDPEAVRDEKVKVLKAFDKIDVNDVLLGQYAKSEDGSKPGYLDDSTVKPNSKAVTYAAFRVNIHNERWDGVPIVLRAGKALDEGKVEIRIQFKPVAKGMFKEIQRNELVIRIQPDEAIYLKINSKIPGISTETSLTDLDLTYSKRYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELEVAWKLFTPLLEAVEKEDEVSLGTYPYGSKGPKELRKYLVDHGYVFNDP GTYQWPLTNTDVKGKI SEQ IDZWF2,atgtcttatgattcattcggtgactacgtcactatcgtcgttttcggtgcttccggtgacttggccagaaaNO: 75 CandidaaaaaaccttccctgccttgtttggcttgtttagagaaaagcaattgcccccaaccgtccagatcattgNucleic Acidgctatgccagatcccatttgtccgacaaggacttcaaaaccaagatctcctcccacttcaagggcggcgacgaaaaaaccaagcaagacttcttgaacttgtgctcttatatgagtgacccatacgacaccgacgacggttacaagaaattggaagccaccgctcaagaatacgaatccaagcacaacgtaaaggtcccagaaagattgttctacttggccttgcctccttccgtcttccacaccgtctgtgagcaagtcaagaagctcgtctaccctaaggacggtaagctcagaatcatcattgaaaagccgtttggccgtgacttggccacctaccgtgaattgcaaaagcaaatctccccattgttcaccgaagacgaagtctacagaatcgaccactacttgggtaaagaaatggtcaagaacttgttggttttgagattcggtaatgaattgttcagtgggatctggaacaacaagcacatcacctcggtgcaaatttccttcaaggaacccttcggtaccgaaggcagaggtggctactttgacaacattggtatcatcagagatgtcatgcaaaaccacttgttgcaagtcttgaccttgttgaccatggaaagaccagtctcttttgacccagaagcagtcagagacgaaaaggtcaaagttttgaaagcttttgacaacattgacgtcaatgacgttcttttgggacaatacgccaagtccgaggatggctccaagccaggttacttggatgactccaccgtcaagccaaactccaaggctgtcacctacgccgctttcagagtcaacatccacaacgaaagatgggacggtgttccaattgttttgagagccggtaaggctttagacgaaggtaaagttgaaattagaatccaattcaagccagtcgctaaaggtatgtttaaggaaatccaaagaaacgaattggttattagaatccaaccagacgaagccatctacttgaagatcaactccaagatcccaggtatctccaccgaaacctccttgaccgacttggacttgacttactccaagcgttactccaaagacttctggatcccagaagcatacgaagccttgatcagagactgttacttgggcaaccactccaactttgtcagagacgatgaattggaagttgcttggaagctcttcaccccattgttggaagccgttgaaaaagaagacgaagtcagcttgggaacctacccatacggatccaagggtcctaaagaattgagaaagtacttggtcgaccacggttacgtcttcaacgacccaggtacttaccaatggccattgaccaacaccgatgtcaaaggtaagatctaa SEQ ID ZWF2,MSYDSFGDYVTIVVFGASGDLARKKTFPALFGLFREKQLPPTVQIIGY NO: 76 CandidaARSHLSDKDFKTKISSHFKGGDEKTKQDFLNLCSYMSDPYDTDDGYK (Amino AcidKLEATAQEYESKHNVKVPERLFYLALPPSVFHTVCEQVKKLVYPKDG Seq.)KLRIIIEKPFGRDLATYRELQKQISPLFTEDEVYRIDHYLGKEMVKNLLVLRFGNELFSGIWNNKHITSVQISFKEPFGTEGRGGYFDNIGIIRDVMQNHLLQVLTLLTMERPVSFDPEAVRDEKVKVLKAFDNIDVNDVLLGQYAKSEDGSKPGYLDDSTVKPNSKAVTYAAFRVNIHNERWDGVPIVLRAGKALDEGKVEIRIQFKPVAKGMFKEIQRNELVIRIQPDEAIYLKINSKIPGISTETSLTDLDLTYSKRYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELEVAWKLFTPLLEAVEKEDEVSLGTYPYGSKGPKELRKYLVDHGYVFN DPGTYQWPLTNTDVKGKISEQ ID PGI1,atgtccactttcaagttagccaccgaattgccagaatggaaaaaattggaacaaacctacaagtc NO: 77CandidacgtgggtgaaaagttcagcgtcagagatgccttcgctaacgacaagaacagattcgaagagttctNucleic Acidcctggatctaccaaaactacgacgactccaagatcttgtttgatttctccaagaacttggtcaacaaggagatcttggaccaattgatcaccttggccaaagaagctggtgtcgagaaattgagagacgctatgtttgctggtgatcacatcaacaccaccgaagacagagccgtgtaccacgttgccttgagaaaccgtgccttgagaaaaatgccagtcgacggtaaggacaccgccaaggaagttgacgacgtcttgcaacacatgaaggaattctccgactccatcagagacggctcttggaccggttacactggtaaggccatcaccgatgttgtcaacattggtattggtggttccgacttgggtccagtcatggttactgaagccttgaaggcctacagcaagccaggcttgaacgtccactttatctccaacattgacggtacccacacccacgaaaccttgaagaacttgaacccagaaaccaccttgttcttggttgcttccaagaccttcaccaccgctgaaaccatcaccaacgccacctccgccaaaaactggttcttagctgccgctaaggatccaaagcacattgccaagcatttcgctgctttgtccaccaacgaagctgaggttgaaaaattcggtatcgacgtcaagaacatgtttggtttcgaaagttgggtcggtggtcgttactctgtctggtccgccattggtttgtctgtcgccatctacattggtttcgaaaacttcaacgacttcttgaagggtggtgaagccatggaccaacactttttgaccactcctttggaaaacaacatcccagtcattggtggtttgttgtctgtctggtacaacaacttctttggtgctcaaacccacttggttgttccattcgaccaatacttgcacagattcccagcttacttgcaacaattgtctatggaatccaacggtaagtctgtcactagagccaacgtcttcaccaactaccaaaccggtaccatcttgtttggtgagccagccaccaacgcccaacactccttcttccaattggtgcaccaaggtaccaagttgatcccagctgacttcatcttggctgctcaatcccacaacccaattgaaaacaacttgcaccaaaagatgttggcctccaacttctttgctcaatccgaagctttgatggttggtaaggacgaagccaaggttaaagccgaaggtgccactggtggtttggttccacacaaggaattctctggtaacagaccaaccacctccatcttggctcaaaagatcaccccagccgctttgggttctttgattgcctactacgaacacgttactttcaccgaaggtgctatctggaacatcaactctttcgaccaatggggtgttgaattgggtaaggttttggctaaggtcattggtaaggaattggatgacaagtccgctgttgttacccacgatgcctccaccaacggtttgatcaaccaattcaagaaatgggaagcttga SEQ ID PGI1,MSTFKLATELPEWKKLEQTYKSVGEKFSVRDAFANDKNRFEEFSWIY NO: 78 CandidaQNYDDSKILFDFSKNLVNKEILDQLITLAKEAGVEKLRDAMFAGDHINT (Amino AcidTEDRAVYHVALRNRALRKMPVDGKDTAKEVDDVLQHMKEFSDSIRD Seq.)GSWTGYTGKAITDVVNIGIGGSDLGPVMVTEALKAYSKPGLNVHFISNIDGTHTHETLKNLNPETTLFLVASKTFTTAETITNATSAKNWFLAAAKDPKHIAKHFAALSTNEAEVEKFGIDVKNMFGFESVVVGGRYSVWSAIGLSVAIYIGFENFNDFLKGGEAMDQHFLTTPLENNIPVIGGLLSVWYNNFFGAQTHLVVPFDQYLHRFPAYLQQLSMESNGKSVTRANVFTNYQTGTILFGEPATNAQHSFFQLVHQGTKLIPADFILAAQSHNPIENNLHQKMLASNFFAQSEALMVGKDEAKVKAEGATGGLVPHKEFSGNRPTTSILAQKITPAALGSLIAYYEHVTFTEGAIWNINSFDQWGVELGKVLAKVIGKELDDKSAVVTHDASTNGLINQFKKWEA SEQ ID ACS2A,atgccagcattattcaaagattctgcccaacacatacttgacaccatcaagtctgaactcccacttgNO: 79 ATCC20336atcccctcaaaaccgcatatgctgtgccgcttgaaaattcagccgaaccaggctactctgccatct(Nucleic Acidacagaaacaaatactccatcgataagttaattgataccccataccccggcttggacaccttgtaca Seq.)agttgtttgaggttgccactgaagcatacggtgataaaccatgtcttggtgccagagtcaagaacgccgatggcacctttggagaatacaagttccaagactacaacaccattcaccaaagaagaaacaacttcgggtcaggtattttctttgtcttacagaacaacccatacaagaccgattctgaagcccactccaagttgaagtacgacccaacaagcaaggattccttcatcttgacaatcttcagtcacaaccgtcctgaatgggccttgtgtgatttgaccagtattgcctattccatcaccaacaccgctttgtacgacactttgggtcccgacaccagtaagtacattttgggtttgactgagtcgccaattgtcatctgttccaaggataagattagaggtcttattgacttgaagaagaacaacccagacgaattgtccaacttgattgttttagtgtccatggatgacttgaccaccgctgatgcctctttgaagaactacggtagtgaacacaacgtcactgtttttgacatcaagcaggttgagaaattgggtgagatcaacccattggacccaatcgagccaaccccagacaccaatttcaccattactttcacctctggtaccaccggtgctaacccaaagggtgtcttgttgaaccacagaaacgctgtcgctggtgtcacttttgtgctcagtagatacgatggccatttcaacccaacagcttattctttccttcccttggcccacatttacgaaagagctagcatccagtttgcattgactatcggttccgccattggattcccacaaggtccatctccattgactttgattgaagatgccaaggtgttgcaaccagacggtttggctttggttcctagagtcttgaccaagttggaagctgccatcagggcacaaactgtcaacaatgacgaaaaaccattggttaaatcggtctttggagctgctatcaacgccaagatggaagcacaaatgaaagaagaaaacgaaaacttcaacccaagtttcattgtctatgatcgtttgttgaacttgttgagaaagaaggttggtttgcaaaaagttacccaaatcagtaccggaagtgctccaatctctccatcaactattcaattcttgaaagcttccttgaatgtcggtatcttgcaaggttacggtttgagtgaatcatttgctggatgtatggcttcttccaaattcgaaccagcggcagccacttgtggtcctactggtgtcaccactgaagtcaagttgaaggatcttgaagaaatgggttacacttccaaggatgaaggtggtccaagaggtgagttattattgagaggtccacaaatcttcaagggatatttcaagaaccctgaggaaactgccaaggccattgacgaagatggttggttccatactggtgatgttgccaagatcaacgacaaaggcagaatttccatcattgatagagcaaagaatttcttcaaattggctcaaggtgaatacgttaccccagagaaaatcgaaggtttgtacttgtccaagttcccatacattgcccaattatttgtccatggtgactctaaggaatcgtacttggttggtgttgtcggattagacccagttgctggtaagcagtacatggagtcgagattccacgacaagatcatcaaggaagaggacgttgttgagttcttcaagtccccaagaaacagaaagatcttagtgcaagacatgaacaagctgattgctgaccaattgcaaggttttgagaagttgcacaacatctacgttgactttgacccattgacggtcgaaagaggcgtcattactccaaccatgaagatcagaagaccacttgctgccaagttcttccaggatcaaatcgatgctatgtacagcgaaggatcattggttagaaatggttctttgtag SEQ ID ACS2A,MPALFKDSAQHILDTIKSELPLDPLKTAYAVPLENSAEPGYSAIYRNKYSID NO: 80 ATCC20336KLIDTPYPGLDTLYKLFEVATEAYGDKPCLGARVKNADGTFGEYKFQDYNTI (Amino AcidHQRRNNFGSGIFFVLQNNPYKTDSEAHSKLKYDPTSKDSFILTIFSHNRPEW Seq.)ALCDLTSIAYSITNTALYDTLGPDTSKYILGLTESPIVICSKDKIRGLIDLKKNNPDELSNLIVLVSMDDLTTADASLKNYGSEHNVTVFDIKQVEKLGEINPLDPIEPTPDTNFTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGHFNPTAYSFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGLALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQMKEENENFNPSFIVYDRLLNLLRKKVGLQKVTQISTGSAPISPSTIQFLKASLNVGILQGYGLSESFAGCMASSKFEPAAATCGPTGVTTEVKLKDLEEMGYTSKDEGGPRGELLLRGPQIFKGYFKNPEETAKAIDEDGWFHTGDVAKINDKGRISIIDRAKNFFKLAQGEYVTPEKIEGLYLSKFPYIAQLFVHGDSKESYLVGVVGLDPVAGKQYMESRFHDKIIKEEDVVEFFKSPRNRKILVQDMNKSIADQLQGFEKLENIYVDFDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRNGSL* SEQ ID ACS2B,atgacatcgacacagagtatcttctccggcgagaagtacactaaggaagaagcgttagcacaat NO: 81ATCC20336tacctttcgggagtgctgttgagaatgctgtcgctataaacgagccagttacaaaccccaagtattct(Nucleic Acidgcaatcttcagaaatgcagcccatctcgaccgcatggttcagaacgtgcaccctgatttaaacacc Seq.)cactacaagctcttcaacaatgctgctgagatgtaccgtgaccgtccatgtcttggtaagcgtccatacaactacaccacacaccaactggatgattacttcctgcactggacgtatggtgaggtctttacgaagaagaataacattggtgctgggtttattcgcgcgttattggagaaccctttccttgacgtgacgttggagtcgcataggaagattgtcaatcatttacgtgactggcccactttcggcatcaacaaatcaccaagggagaacttgaactatgagattgagaagaattgttcgttcattttgactatttttgccgtcaatcgtgctgaatggatcttgactgatttggcatgcagttcgtatgcaatcacaaacaccgcgttgtatgatacattaggtcccgacgtgtctcagtatatcttgaacttgactgaatcaccaattgtcgtttgcacccatgacaagatccaggttttgttgaacttgaaaagaaagtaccctgagcagaccaagaacttgatttctatcgtgtcaatggacccaattgatttagtcacgcagggaacaatcgaacaagcttatgagttgggggtcacaattcaaggtttgaatcagattgagaaaattggtgttctgaacccaattcaacaattggaaactggtacagaagctttgtttaccatttcattcacttcaggaactactggtagtaaacccaaaggagtgatgatttctcaaggtggtgctgctgcctacgtcacgtgggggttgagctgttgtccacaggctaaacctggtgataaggcgtatattttcttgccgttgactcatttgtatgaaagagaaacttgtgcgtttgcctatagttctgggtactatttggggttcccgcagattaacctaggcaaaaagaaggtgaatccttttgagaacatgcttaacgatttgagaatcttcaaaccaacatatatgtccatggttcctagattattgaccaggttggaagcgttgatcaagagtaagatcaaggagttgccacaagctgaccaggacagagtgaatggtataattgagatcaagataagggagcaaagcaaggctgacggcgccaaagggtttgacgcaaccttggacaacgaccctacctacaaatcattagctaagtttgttgggtatgaaaacatgagatgggttcagactgcaagtgcaccaattgcccccaccacacttgtctacttgaaagcgtctttgaacatcggtgccagacaacaatacgggttgactgaaagtggggccgccatcacaagtaccggggagtacgaagcatccccaggaggttgtggtgttgtattaccaactgggcagtgccgactctactccgtttccgagatggggtactccttggacaaattagaaggcgaggtgttgctccagggcccacagatgttcaaagggtactactacaactacgaggaaaccgagaatgcagttactgaagacggatggttccattcaggagacattgctcgggttgaccccgcgacaggtcgcctcgacataattgaccgagtcaagcatttcttcaaattggctcagggagagtacatctccccagagcgtatcgagaacaggtacttgtcgtcgaacccagacatctgccagctctgggtgcacggggactctaaggagcactacttgattggcatagtgggggtggagtacgaaaaaggattgaagtttatcaatgaggagtttggatataacaagattgacatgcagccggatgatttgttggacattttgaactcggcggaggtgaaggcacggttcttgagcaagctaaatagactggttaaagataagttgaacgggtttgagatcttgcataatatctttattgagtttgagccgttgacggtccagagagaagttgttactccgacattcaaaattagaagaccaatctgtcgtaagttcttcaaggcccagcttgatgcgatgtacgccgaggggtccttaatcagcgctgccaagttgtag SEQ ID ACS2B,MTSTQSIFSGEKYTKEEALAQLPFGSAVENAVAINEPVTNPKYSAIFR NO: 82 ATCC20336NAAHLDRMVQNVHPDLNTHYKLFNNAAEMYRDRPCLGKRPYNYTTH (Amino AcidQSDDYFSHWTYGEVFTKKNNIGAGFIRALLENPFLDVTLESHRKIVNH Seq.)LRDWPTFGINKSPRENLNYEIEKNCSFILTIFAVNRAEWILTDLACSSYAITNTALYDTLGPDVSQYILNLTESPIVVCTHDKIQVLLNLKRKYPEQTKNLISIVSMDPIDLVTQGTIEQAYELGVTIQGLNQIEKIGVSNPIQQLETGTEALFTISFTSGTTGSKPKGVMISQGGAAAYVTWGLSCCPQAKPGDKAYIFLPLTHLYERETCAFAYSSGYYLGFPQINLGKKKVNPFENMLNDLRIFKPTYMSMVPRLLTRLEALIKSKIKELPQADQDRVNGIIEIKIREQSKADGAKGFDATLDNDPTYKSLAKFVGYENMRWVQTASAPIAPTTLVYLKASLNIGARQQYGLTESGAAITSTGEYEASPGGCGVVLPTGQCRLYSVSEMGYSLDKLEGEVLLQGPQMFKGYYYNYEETENAVTEDGWFHSGDIARVDPATGRLDIIDRVKHFFKLAQGEYISPERIENRYLSSNPDICQLWVHGDSKEHYLIGIVGVEYEKGLKFINEEFGYNKIDMQPDDLLDILNSAEVKARFLSKLNRSVKDKLNGFEILHNIFIEFEPLTVQREVVTPTFKIRRPICRKFFKAQLDAMYAEGSLISAAKL* SEQ ID ACS2C,atgtccaccttattcaacgagccacctgaacagatctaccagagtctcttgactcagtacaacaacNO: 83 ATCC20336ccactcgattacgcatccagtgttgcactcccaaatacgcaagaaccgggatattcatctatctaca(Nucleic AcidggaatgtgtttgatcccagtaagcttgttacctgtccacatcccgagttagacacgttgtacaaaattttSeq.)tgagttcagtgttattgtttacggagacaagccgttccttggccaccggatcaaaaacccagatggtacctttggcgagtacacgtttgaaacttacaagcaggtctatgaaagaagaaacaatcttggttctggtatctactacgtcttggaaaacagtccataccggacctcatccgaagctcatgcgaaattaaagtatgaccctaccaacgacaacccattcattttggcgcttttcagtcataatcgtccagaatgggcattgtgtgatgtcacaaccagtgcttatggtttcatcaacactgcattgtacagcaccttgggtccagataccagcagatacatcttgtctgtcaccgattgtccaattgtagttgccacgaaggacaagattgaagggttgatcaacttgaagaagaaaaatcccaaagacttggtcaacttgattgttcttgtttcattagatgaacttaccgttgaagacgataagttgagatcattgggtcgtgaaaacaacattgtggtttatgctttgaaggaagttgaaagactcggtgcagctaacccattggcaccaattgctccaaccccagacactgtcttcacgatttcgtttacttcgggtaccagcggagcagcacctaaaggtgttgtgttgacaaatagaatcttggcttgcggaatagcatcccattgttctctcgttgggtttggtcctgaccgtgtcgagtacagtttcttgcctttggctcatatctacgagagaatggtgcttcagtttggaataatagctggagtgaagatcgggtatccacagggtccttctccaacaaccttgtttgaagacatcaaggttttgcaaccaacgatgttgtgtttggttcccagagtattcacgaagatagaagctgccatcaaggcccagacagttgaaaatgaatcagatccagaactcagagctaagtttatcgaaataatcaacaagaaagtggagttgcaacaacaacaggactttacaaatccaagtctcccagaaggtgacaagctcttacaacaattgcgtgagacccttggaatgggtaaacttcaattcatgaacactggatcagctccactgtcagaggagtcttatcgttttatccaagctgtgatgaacatgcctaatgggttccgttgtggttacggtttgacagaaagcgctgctggtcttgcaatctccccaccatacgccaatgaattctcatgtgggcccatttcccagaccaccgagtttagattgaaagatcttgttgatatgggttacacttcgaaagataaagagggagtaaggggtgaattgttattgagaggtcctcaaatcttctcttactactacaagaacccagaggagacggccaaggctatagacaaggacggatggttccatactggtgatgtggcttctctcaccgaagcacatggaaacaggtttcagattatcgacagagccaagaacttcttcaagttgtcccaaggagagtatgtgtctccagagaagattgaaaatgtgtacatggctcagtttccgttcatctcgcagttgtttgttcatggagattcattggagtcatatttggttggtgtcgttggaatcgacaaagcattggttgatccttatttgaagaaacgcttcaacgtggagttggacacaccagctgagatcttcaagttttttgagaacccacggaacagaaagacattgttgcaagacatgaacaaggcggttggtagtgagttgcaaggattcgaaaaattgcataatgtttttgttgattttgaaccattgacccttgagagaggagttattacgccaactgtcaagatcagaagagctaactgtgtcaacttctttaagcagcacatccaaagtatgtatggcgaaggatctttgctcaagaatagtaacttatagSEQ ID ACS2C, MSTLFNEPPEQIYQSLLTQYNNPLDYASSVALPNTQEPGYSSIYRNVF NO: 84ATCC20336 DPSKLVTCPHPELDTLYKIFEFSVIVYGDKPFLGHRIKNPDGTFGEYTF (Amino AcidETYKQVYERRNNLGSGIYYVLENSPYRTSSEAHAKLKYDPTNDNPFIL Seq.)ALFSHNRPEWALCDVTTSAYGFINTALYSTLGPDTSRYILSVTDCPIVVATKDKIEGLINLKKKNPKDLVNLIVLVSLDELTVEDDKLRSLGRENNIVVYALKEVERLGAANPLAPIAPTPDTVFTISFTSGTSGAAPKGVVLTNRILACGIASHCSLVGFGPDRVEYSFLPLAHIYERMVLQFGIIAGVKIGYPQGPSPTTLFEDIKVLQPTMLCLVPRVFTKIEAAIKAQTVENESDPELRAKFIEIINKKVELQQQQDFTNPSLPEGDKLLQQLRETLGMGKLQFMNTGSAPSSEESYRFIQAVMNMPNGFRCGYGLTESAAGLAISPPYANEFSCGPISQTTEFRLKDLVDMGYTSKDKEGVRGELLLRGPQIFSYYYKNPEETAKAIDKDGWFHTGDVASLTEAHGNRFQIIDRAKNFFKLSQGEYVSPEKIENVYMAQFPFISQLFVHGDSLESYLVGVVGIDKALVDPYLKKRFNVELDTPAEIFKFFENPRNRKTLLQDMNKAVGSELQGFEKLHNVFVDFEPLTLERGVITPTVKIRRANCandidaNFFKQHIQSMYGEGSLLKNSNL* SEQ ID CAT2,atgtttaactttaagttgtcgcaacaagtattaaagaattccaccaaatccattatgccaattttgaaaNO: 85 ATCC20336aaaccattctccaccagccacgcaaagggtgacttgttcaaataccagtcacaattacccaagttg(Nucleic AcidcctgttcctactttggaagaaaccgcatccaagtacctcaagaccgttgagccattcttgaaccaagSeq.) agcaattggaatccaccaaggccaaagtcgctgagtttgttagaccaggtggtgccggtgaagccttgcaagccagattgaacaactttgccgccgacaaggacaactggttggctgaattttgggacgactatgcatacatgtcttatagagatcctgttgttccatatgtttcttactttttcagtcacaaggatgtcaagaacatcattggccaagaccaattgttgaaggccactttgattgcttactacactattgagttccaagaaaaggttttggacgaaagtttggacccagaagtcatcaagggtaacccattctgtatgaacgccttcaagtacatgttcaacaactcgagagttccagctgaaggctccgacatcacccaacactacaacggtgaagaaaaccaatttttcgttgtcatctacaagaacaacttctacaaggttccaacccacaagaacggccaaagattgaccaagggtgaaatctacagctacttgcaagaaatcaagaacgatgccactccaaagggtctcggtttgggtgctttgacctcattgaacagagacgaatggttgagtgcctacaacaacttgttgaagtccccaatcaacgaagcttccttgggatccatctttgcttccagctttgtcattgccttggactccaacaacccagtcaccattgaagaaaaatccaagaactgctggcacggggacggtcaaaacagattctttgacaagcctttggaattcttcgtcagtgctaacggtaactctggtttccttggtgaacactccagaatggacgctaccccaaccgtgcaattgaacaacaccatctacaagcaaatcttggaaaccaatccaaacgacttgattgttgaaattggttcttctgctccaagattcggcaatgctgaaatcttgcctttcgacatcaacccaaccaccagagccaacatcaaagacgctattgccaagtttgacgccaccattgctgcccacgacgaagaaatcttccaacactacggttacggtaagggattgatcaagaagttcaaggtctccccagatgcctacgtgcaattgttgatgcaattggcatacttcaagtacaccggcaagatcagaccaacttatgaatccgccgccaccagaaagttcttgaagggtagaaccgaaaccggtagaactgtctccaacgaatccaagaagtttgttgagacctggtccgatccaaaggccagcagcgccgacaaggttgccactttccaagctgctgctaagcaacacgttgcctatttgtctgctgctgccgatggtaagggtgttgaccgtcacttgtttggtttgaagcaaatgattcaaccaggcgaaccaatccctgaaatcttcactgacccaatcttcagctattctcaaacctggtacatttcttcttcccaagttccatctgaattcttccaatcttggggttggtcgcaagtcattgatgacggtttcggtttggcttacttgatcaacaacgactggatccacgttcacatttcttgtaagagaggcaacggcttgcaatctgaccacttgaaatggtacttggttgaaagtgctaatgaaatgaaggatgttttgactaagggattattgactgatgctaagcctaagttgtaa SEQ ID CAT2,MFNFKLSQQVLKNSTKSIMPILKKPFSTSHAKGDLFKYQSQLPKLPVP NO: 86 ATCC20336TLEETASKYLKTVEPFLNQEQLESTKAKVAEFVRPGGAGEALQARLN (Amino AcidNFAADKDNWLAEFWDDYAYMSYRDPVVPYVSYFFSHKDVKNIIGQD Seq.)QLLKATLIAYYTIEFQEKVLDESLDPEVIKGNPFCMNAFKYMFNNSRVPAEGSDITQHYNGEENQFFVVIYKNNFYKVPTHKNGQRLTKGEIYSYLQEIKNDATPKGLGLGALTSLNRDEWLSAYNNLLKSPINEASLGSIFASSFVIALDSNNPVTIEEKSKNCWHGDGQNRFFDKPLEFFVSANGNSGFLGEHSRMDATPTVQLNNTIYKQILETNPNDLIVEIGSSAPRFGNAEILPFDINPTTRANIKDAIAKFDATIAAHDEEIFQHYGYGKGLIKKFKVSPDAYVQLLMQLAYFKYTGKIRPTYESAATRKFLKGRTETGRTVSNESKKFVETWSDPKASSADKVATFQAAAKQHVAYLSAAADGKGVDRHLFGLKQMIQPGEPIPEIFTDPIFSYSQTWYISSSQVPSEFFQSWGWSQVIDDGFGLAYLINNDWIHVHISCKRGNGLQSDHLKWYLVESANEMKDVLTKGLLT DAKPKL* SEQ IDCROT (Amino MPQEDYSDIVSEATEFVSSSLINLIQRHLEAVAEREEFSNYLNVVNND NO: 87Acid Seq.) MTPSIYGEIRGDILPRNPYLILEEDPYSKTINPPNQAQRAANLINSSLKFIITLRNETLKPDVTPKHGNPLTMKCYRNLFGTTRIPEFEEHNHDIKMRKYEHINDSRHILIIANNQFYTLEVITEYTEEEYQETKSKHKIWFNDHELSLILQQIIDESKKVDAVKSINNSIGSLTTQTLKHWKLARLELEQSNLENIKKIDDALFVVILDSNAPETDGEKTAVISHGTSVLSADNVQVGTCTSRWYDKLQLIVTQNSVAGVVWESMSMDSTAILRFISDIYTDSVLKLAKNINGSEYTLFDSNIVFASSSISKPEAVMIYFNKTKELQNIIHLSETRLADLINQHEYMTHRIKLDSYLTSKFNLSVDSIMQVCFQIAYYSLYGRVVNTLEPITTRKFKDARTELIPVQNEVLGNLVKLYITSASALEKFEAFKKCCELHTHQYHDAMIGKGFERHLMTIIQVIKKPKAVVRLNELNSHLPPIPDLTKEPVTIPLLLNPAIDKLLSPELLISNCGNPALRLFGIPPAIDQGFGIGYIIHRDKVLITVCSKHRQTERFLDTFHRVVRDLKVNLRQKSNFLXXXXSVTRNKENTSC RGCESSMS SEQ ID CROTatgccgcaggaggactactcagacatcgtgagcgaggccaccgagtttgtctctagcagcttgat NO: 88(Nucleic Acidcaacttgatccagcggcacttggaagctgttgctgagagagaggagttctcaaactacttgaacgt Seq.)cgtcaacaatgacatgactccgtccatctacggtgagattaggggtgacatattgcccagaaacccgtaccttatcttggaagaagacccgtacagcaagacgatcaacccaccgaaccaggcccagagagcagccaacttgatcaactcgagtttgaagttcattattacgttgaggaacgagacgttgaagccagatgtcactccaaagcacggaaacccgttgacgatgaagtgctacaggaacttgtttggcacgacgaggatcccggaatttgaagagcacaaccacgacatcaagatgagaaagtacgaacacatcaacgactcacgacacattttgattatcgccaacaaccagttctacacattggaagtgatcacggagtacacagaggaggagtaccaggagaccaagtccaagcacaagatctggttcaatgaccatgagctttcgttgatcttgcagcaaatcatagacgagtccaagaaggttgacgccgtcaagtccatcaacaactccattgggtcgttgaccacgcagacgttgaagcattggaagttggctaggttggagttggagcagtcgaatctggaaaacatcaagaagattgacgatgcgttgtttgtcgtgattttggactctaacgcaccagaaactgacggagagaaaacggcggtgatttcccacggtacgtccgtgttgtcagcggacaacgtgcaggttggtacctgtacctcgcgttggtacgataagttgcagttgattgtcacccagaactccgttgctggtgtggtgtgggaatccatgtcgatggacagtactgctattttgagattcatcagtgatatctacaccgactcggtgttgaagttggccaagaacatcaacgggtccgagtacactttgtttgactccaatattgtgtttgcgtcttcttcaatcagcaagccggaagccgtcatgatctatttcaacaaaacaaaggagttgcagaacattatccatctttcggaaaccagattggctgacttgatcaaccaacacgagtacatgacgcaccgtatcaagttggattcgtacttgacgagcaagtttaacctttccgtggactccatcatgcaggtgtgtttccagattgcttattactcgttgtacggtagagttgtcaacacgttggagccaatcaccaccagaaaattcaaggacgccagaaccgagttgatcccggttcagaatgaagttcttggcaacttggtcaagctctacatcaccagcgccagtgcactggagaagtttgaagcattcaagaaatgttgtgagttgcacacccaccagtaccacgatgccatgattggtaaaggtttcgaaagacacttgatgacgatcatccaagtgatcaagaaaccgaaagctgtggtcagattaaacgaacttaacagccacttgccgccaattcctgacttgaccaaggaaccagtgaccattccgttattgctaaacccagccattgacaagttactgagccccgagttgttgatttccaactgcggtaatcctgcattgagattgtttggtatcccgccagccatcgaccaagggtttggtattgggtacattatccaccgcgacaaggtgttgatcactgtttgttccaagcacagacaaacggaaaggttcttggacactttccaccgtgttgttcgtgatttgaaggtcaacttgagacaaaagagcaactttttgnnnnnnnnnngatcagtgactcggaacaaagaaaacacgagttgcagaggttgcgaatcgagcatgagt SEQ ID FAT1,atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattNO: 89 ATCC20336gccgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccag(Nucleic Acidcagaggtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaag Seq.)cgttggcgttcccaagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgacgatcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttgaagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattgtcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagccattgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcagagataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttgtttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaaccgatactgactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcatgtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagcagaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctgtaccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcagaaatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaaaggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcgacagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattgatgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggtaaagccaaattgtag SEQ ID FAT1,MSGLEIAAAAILGSQLLEAKYLIADDVSLAKTVAVNALPYLWKASRGKA NO: 90 ATCC20336SYWYFFEQSVFKNPNNKALAFPRPRKNAPTPKTDAEGFQIYDDQFDL (Amino AcidEEYTYKELYDMVLKYSYILKNEYGVTANDTIGVSCMNKPLFIVLWLAL Seq.)WNIGALPAFLNFNTKDKPLIHCLKIVNASQVFVDPDCDSPIRDTEAQIREELPHVQINYIDEFALFDRLRLKSTPKHRAEDKTRRPTDTDSSACALIYTSGTTGLPKAGIMSWRKAFMASVFFGHIMKIDSKSNVLTAMPLYHSTAAMLGLCPTLIVGGCandidaSVSQKFSATSFWTQARLCGATHVQYVGEVCRYLLNSKPHPDQDRHNVRIAYGNGLRPDIWSEFKRRFHIEGIGEFYAATESPIATTNLQYGEYGVGACRKYGSLISLLLSTQQKLAKMDPEDESEIYKDPKTGFCTEAAYNEPGELLMRILNPNDVQKSFQGYYGNKSATNSKILTNVFKKGDAWYRSGDLLKMDEDKLLYFVDRLGDTFRWKSENVSATEVENELMGSKALKQSVVVGVKVPNHEGRACFAVCEAKDELSHEEILKLIHSHVTKSLPVYAQPAFIKIGTIEASHNHKVPKNQFKNQKLPKGEDGKDLIYWLNGDKYQELTEDDWSLICTGKAKL* SEQ ID PXA1,atggtcaacatatcgaaattgacgggttataacaagcaggacatcaggaatgtggtgctattgctaNO: 91 ATCC20336caggagtttgtcaagacctacaaagacaacaagatcaaactcaactacctgagtagacctgtcat(Nucleic AcidcttgttcttgagtaccttggttgcaactgccggtattggggtgtttttcaccttgagaagcatcgtcactaSeq.) agtacaacgagtacctactcaacaagagattgagacgcccaagtcttatcagacaatcctccaatatcttgaagaacggatcccgtgagatctttatccagaagggcaacggcaaagtaacaagaatcatcatcccaaaagcaaacaacgaccagtatgccgccgacaagtatttgtataaagattttgcccgcaacgagcaaatattgcaacagcaaaagggaaggctcttcaattccagattcttgaaccagttgaccattatctggaagatcttgattccaaagttctactgccaaaacacttccttgttgttatcgcagtgcttctttttgattttcagaacatggttgtccttgttgattgccaagctagatggtcagattgtcaagaacttgattgctgcagacggtaggaagtttgcccgtgacttgatttactttttgttgattgccttccctgcttcgtacaccaacgccgctatcaaatacttggaattgagattggcgttaggattcagaactaatcttaccagatacatccatgacatgtacttggacaaaaccatgtcgtactacaaagtgggattgaacggcgccgatatccaaaacatagaccagtacatcaccgaagatgtcaccaaattctgtatgtcgttgtgttcgttgttttcctccatgggtaagccattcattgacttgatctttttcagtgtttatttgagagacaatttgggtactggtgccattattggcatttttgccaactattttgctaccgccatcatgttgaaaaaggcaacaccaagattcggtaagttggctgccaaaagaacccacttggaaggtgtttatttcaaccaacagttgaacataatgaccaacagtgaagagattgggttctacaaaggatcgaagattgagaagtccaagcttgcggagaactttgacaagttgatgggtcacgtatcgagagaaatcaatttatcgtccagctatgccgctctagaagactacgtgcttaaatacacgtggctggcctggggttacatcttttctggtctacctgtgtttttggatgtgcttttccctaaagaagacccaagtagtggccatattgctgatatagatgacgatgaccatgcccatggacacgggcacaccggggaagagaccagctcaacaactgaaaacatgaagaccttcgtcaccaacaagcgattattgttgagtcttgccgatgctggttccagattgatggttagtttgaaagaggtcaccacgttgacaggtataacgaatagagtcttcaacatgttgactcagctccaccgtgtccatgatcctaaatttgactacggtgacaagtatggtttgcctgatattcacgggacttatcaattgaactacgatggtttgagattggaacatgttccaattactgtgccaactgccgagggttcttactccacaccattgatcccagacctcacttttgacatcaagggcaagaatttgttatttgttggtccaaacggttcgggcaaaacttctgttgccagggttcttgcaggtctttggcccttgtatgccgggttagtgctgaaaccactggatttgttctttaacccacaaaagagttatttcaccaccggaagtttgcgtgaccaagttgtttaccctaatagatccgaaaacaccaccaacgatcaaattttccacatcttacactgtgtacacttagaccatattgttaaacggtacggattgaaccagaacttggatttcgctaaaacattgagtggaggtgagaagcaaagattgagtttcgccagagtgttgtttaacagaccaagtattgtcattcttgatgattcgacgtcggcgttgtccccagatatggaagagttgatgtaccaggtgttgcaagatcacaagatcaattacgtcacactttcaaatcgtccctctctcagtaagttccatgataaagtatttgaaatataa SEQ ID PXA1,MVNISKLTGYNKQDIRNVVLLLQEFVKTYKDNKIKLNYSSRPVILFLSTL NO: 92 ATCC20336VATAGIGVFFTLRSIVTKYNEYLLNKRLRRPSLIRQSSNILKNGSREIFI (Amino AcidQKGNGKVTRIIIPKANNDQYAADKYLYKDFARNEQILQQQKGRLFNSR Seq.)FLNQLTIIWKILIPKFYCQNTSLLLSQCFFLIFRTWLSLLIAKLDGQIVKNLIAADGRKFARDLIYFLLIAFPASYTNAAIKYLELRLALGFRTNLTRYIHDMYLDKTMSYYKVGLNGADIQNIDQYITEDVTKFCMSLCSLFSSMGKPFIDLIFFSVYLRDNLGTGAIIGIFANYFATAIMLKKATPRFGKLAAKRTHLEGVYFNQQLNIMTNSEEIGFYKGSKIEKSKLAENFDKLMGHVSREINLSSSYAALEDYVLKYTWSAWGYIFSGLPVFLDVLFPKEDPSSGHIADIDDDDHAHGHGHTGEETSSTTENMKTFVTNKRLLLSLADAGSRLMVSLKEVTTLTGITNRVFNMLTQLHRVHDPKFDYGDKYGLPDIHGTYQLNYDGLRLEHVPITVPTAEGSYSTPLIPDLTFDIKGKNLLFVGPNGSGKTSVARVLAGLWPLYAGLVSKPSDLFFNPQKSYFTTGSLRDQVVYPNRSENTTNDQIFHILHCandidaHLDHIVKRYGLNQNLDFAKTLSGGEKQRLSFARVLFNRPSIVILDDSTSALSPDMEELMYQVLQDHKINYVTLSNRPSLSKFH DKVFEI* SEQ ID PXA2,atgacagtggagaatgcaaaactacagaagaactcgttggcggttctgctcttgaaggtgtacaa NO: 93ATCC20336atccaacagatcattattgttaaacacctcatacatcatattaatcattgctgccttcactggcgcaac(Nucleic Acidgaataccgggcgaggcacctcctccagatcatcggcaaaagtagagaccgatgaagaacaat Seq.)cggttaaaaagaaacaccccaagctctctagagagtccttccatagactaagaaaagcaatcttgccaactttctttgatagaactatagtttactttttcgccaacttgactttgttggtggtcagagcattattgacacttagagttgctacccttgacggtcagcttgtgggggcattggtttcaagaagaataagggtgtttgccaagtacttgttgtactggatgcttcttggtatccccgctgctttgacaaatgccttgttgaactggaccaaactgaacttgagcaagagcattagaatgaacttgaataataacatcatggaggaatacttgccagataacttggacccaaactattattcattgatccatttgactgataacaagattagagacccaaatcagagaataaccactgatactagtcgtttgagcgatgccttggcaagcttgcccggtcacatattgaagccaacgttggatatcatattgtgtgcgcaacagttaagcaagagcggtgttggtaatggggaaggtacgttggcattaggtatattggcacacttctcaaccatgatcatccgtttcttctccccgccatttgccaagttggcggctgagagagctaaccttgaaggtcagttgcgttccgcgcattccaagattgttgccaacagtgaagaaattgctttcttgggtggtcatgaccgtgagttggatcacatcgaccactgctactatactttggagagattctcgaaaggcgaatattggaagcgagccatacacgaaatcacacaaacgtttattgtgaagtacttttggggtgttgcaggtttagtgttgtgttctgcaccggttttcattgccaaatacttgggtgagccggaagataagaatgttgctggtaatttcatcaccaacagaagattgttgatgagtgcctcggattccttggatcgtttaatctattctagaagatacttgttgcaagttgtcggtcatgctaccagagtgtctgacttcttggacactttacatgaagtggaggagaagaagaagagaatcacatcgaatgtgcagtttaacaacgacgagattactttcgatcatgttagattgatgactccaacggaagtgaccttgatcccagacttgaacttttccattaaaccaggtgaccatttgttgattgtggggccaaacggttcaggtaagtcgtcgttgttcagaatgttgggtgggttgtggcccgttaggtttggtactattagaattccaaacacagagaacatgttctacttgccgcaaaaggcttaccttgttgaaggatcattcagagagcaaatcatttatccacacaacgtgactcaacagaagaagactgatcaacaattgaaagagatcttgaaggttttgaaattggaagattactcagggcaattggatgaggttaagaaatggagcgaagaattgtccattggtgctcaacaaagattggctatggctagattgtactaccacgaacctaagtttgctgtcttggacgaatgtacttcagctgtgtcaccagacatggaacaactcatgtaccaacacgcacaaggtttgggtatcacgcttttgtccgttgcccatagacctgcattgtggcacttccacaaatacttgttggaattcgacgggaagggtagttactactttggtacgttggatgaaaagcacaaaatgaagttagaagaagaagaacgactcaagaaggagaatgaaaagaagagtgtcgccaagaagtag SEQ ID PXA2,MTVENAKLQKNSLAVSLLKVYKSNRSLLLNTSYIILIIAAFTGATNTGRG NO: 94 ATCC20336TSSRSSAKVETDEEQSVKKKHPKLSRESFHRLRKAILPTFFDRTIVYFF (Amino AcidANLTLLVVRALLTLRVATLDGQLVGALVSRRIRVFAKYLLYWMLLGIPA Seq.)ALTNALLNWTKSNLSKSIRMNLNNNIMEEYLPDNLDPNYYSLIHLTDNKIRDPNQRITTDTSRLSDALASLPGHILKPTLDIILCAQQLSKSGVGNGEGTLALGILAHFSTMIIRFFSPPFAKLAAERANLEGQLRSAHSKIVANSEEIAFLGGHDRELDHIDHCYYTLERFSKGEYWKRAIHEITQTFIVKYFWGVAGLVLCSAPVFIAKYLGEPEDKNVAGNFITNRRLLMSASDSLDRLIYSRRYLLQVVGHATRVSDFLDTLHEVEEKKKRITSNVQFNNDEITFDHVRLMTPTEVTLIPDLNFSIKPGDHLLIVGPNGSGKSSLFRMLGGLWPVRFGTIRIPNTENMFYLPQKAYLVEGSFREQIIYPHNVTQQKKTDQQLKEILKVLKLEDYSGQLDEVKKWSEELSIGAQQRLAMARLYYHEPKFAVLDECTSAVSPDMEQLMYQHAQGLGITLLSVAHRPALWHFHKYLLEFDGKGSYYFGTLDEKHKMKLEEEERLKKENEKKSVAKK* SEQ ID PEX11,atggtcgccgattctttagtctaccacccaaccgtctccaaattagtcaagttcttggacacaaccccNO: 95 ATCC20336aaagagggaaaaggtcttcagattattgtcctacttgtccagattcttgggctactacgcctacagaa(Nucleic Acidagggctactccaaggaaaccatcgcccttttcgccaacttgaaaggaaacttcacattcatcagaa Seq.)aggccatgagattcttgaagccaataaatcacttgcaattggcctccaaggcatacgacaacaagttgttggacccagtcttgcagatcaccaccatcatcagaaacttggcctacgccggctacttgaccatcgacggtgtcatattcttcaagttgttgggtctcattgacgccaagaagttccctaacttggctacatacgcctccagattctggttgatcgggttgattgccggtttgatcaactccttgagaatcatctactccttgaaggactacgagcaccaggagggcgacaaggagaaggagaccgacgctaaggctatccacactaagttgtacgccgctaagagaaaattggtctgggacttgttggatacttttattgctttgaactccttggacatcttgcatttcaccgagggtgacgtcgggttcgctggtactatcacctccctcttgggattggaagacttgtggaaggccacttaa SEQ ID PEX11,MVADSLVYHPTVSKLVKFLDTTPKREKVFRLLSYLSRFLGYYAYRKGY NO: 96 ATCC20336SKETIALFANLKGNFTFIRKAMRFLKPINHLQLASKAYDNKLLDPVLQIT (Amino AcidTIIRNLAYAGYLTIDGVIFFKLLGLIDAKKFPNLATYASRFWLIGLIAGLIN Seq.)SLRIIYSLKDYEHQEGDKEKETDAKAIHTKLYAAKRKLVWDLLDTFIALNSLDILHFTEGDVGFAGTITSLLGLEDLWKAT* SEQ ID UGTA1MFEDRLMAASGLDVPIILEDSPFFKTEMKPSTSYNITLLTIGSRGDVQP NO: 97 (Amino AcidYMALGKGLVKEGHNVTIATHGEFGDWIKKSGLNFKEIAGNPAELMSF Seq.)MVTHNTMSVGFLKDAQKKFKSWIATLLTTSWKACQGSDILIESPSAMAGIHIAEALGIPYFRAFTMPWTRTRAYPHAFFVPDQKKGGSYNYLTHVLFENIFWKGISGQVNKWRVQELDLPKTNLYRLQQTRVPFLYNVSLTVLPPAVDFPDWIKVTGYWFLDEGSGDYKPPEELVKFMSDAAADGKKIVYIGFGSIVVKDAKSLTKAVVEAVKRADVRCILNKGWSDRLDKKGKDDIEVELPPEVYNSGAIPHDWLFPRVDAAVHHGGSGTTGASLRAGTPTIIKPFFGDQFFYATRVEDLGAGLGLKKLTAKTLANALVTVTEDLKIIEKAKRVSEQIKHEHGVLSAIEAlYSELEYSRNLILVKDIYNQNYKRHHPDFRSQSGIQSPVEPSDDEDEEDEEEEDSNEDDDEDNEDYEDESSDQRSKAS SEQ ID UGTA1atgtttgaagacagattgatggctgcgtctggtttggatgttcctattatcttggaagactcgccattctttNO: 98 (Nucleic Acidaagacagaaatgaagccatcaacctcttacaatatcacgttgttgactattgggtcgcgaggtgat Seq.)gtgcaaccatacatggctttaggtaaaggcttagttaaagagggccacaatgttactatcgctactcacggagagttcggagactggatcaagaagagtggattgaactttaaagaaattgctggtaatcctgccgagttgatgtcgtttatggttacccacaacaccatgtctgttggtttccttaaggatgcccagaagaagttcaaatcctggattgccacgttgttgactaccagttggaaggcgtgtcaaggttctgatatcttgattgaaagtccttcagccatggccggtatccacattgccgaagccttaggcattccttatttcagagcattcacgatgccatggactagaactagagcgtacccgcatgcattctttgtgcctgatcaaaagaagggtggttcatacaattatttgacacatgtcttgtttgaaaatatcttctggaaaggtatttctggacaggttaacaagtggagagtgcaagagttggatttgcccaagaccaacttgtacagattgcagcaaacgagagtgccgttcttgtacaatgtctcgctcactgttttgccaccggccgttgacttccccgattggattaaagtcactggttactggtttttggatgaaggttctggtgattacaaacctcctgaagagcttgtcaagtttatgagtgatgccgctgctgatggcaaaaagattgtctacattggatttggttccattgttgttaaggacgccaagtcgttgacgaaagctgttgttgaagctgttaagcgtgccgacgtccgttgtatcttgaacaaaggttggtccgacaggctagacaagaagggtaaggacgatatcgaagttgagttaccgccagaagtgtacaactcgggtgctatccctcatgattggttgttcccacgtgtcgacgcagctgtacaccacggtggttctggtaccactggtgctagtttgcgtgctggtacacctactattatcaaaccattctttggagaccaatttttctacgccacgcgagttgaagatttaggcgccgggttggggttgaaaaaattgactgcaaaaacgttggcaaacgcgcttgttaccgttaccgaagacttgaagattattgaaaaggcaaagagagtcagcgagcaaatcaaacacgagcatggtgttctcagtgccatcgaagctatctactcagagttagagtactccaggaacttgatcttggtgaaggatatctacaaccagaactacaagcgtcaccacccagatttcagatcacaatctggtattcaatcgccagttgagcctagcgatgacgaggacgaagaagacgaagaagaagaagacagcaatgaagacgacgatgaggacaatgaagattatgaagacgagtcatcagaccagcggtcaaaggcttcatag SEQ ID IDP2, S. catgacaaagattaaggtagctaaccccattgtggaaatggacggcgatgagcaaacaagaata NO: 99(Nucleic AcidatctggcatttaatcagggacaagttagtcttgccctatcttgacgttgatttgaagtactacgatctttcSeq.)cgtggagtatcgtgaccagactaatgatcaagtaactgtggattctgccaccgcgactttaaagtatggagtagctgtcaaatgcgcgactattacacccgatgaggcaagggtcgaggaatttcatttgaaaaagatgtggaaatctccaaatggtactattagaaacattttgggtggtacagtgttcagagaacctattattatccctagaattccaaggctagttcctcaatgggagaagcccatcatcattgggagacacgcattcggcgatcagtacaaagctaccgatgtaatagtccctgaagaaggcgagttgaggcttgtttataaatccaagagcggaactcatgatgtagatctgaaggtatttgactacccagaacatggtggggttgccatgatgatgtacaacactacagattcgatcgaagggtttgcgaaggcctcctttgaattggccattgaaaggaagttaccattatattccactactaagaatactattttgaagaagtatgatggtaaattcaaagatgttttcgaagccatgtatgctagaagttataaagagaagtttgaatcccttggcatctggtacgagcaccgtttaattgatgatatggtggcccaaatgttgaaatctaaaggtggatacataattgccatgaaaaattacgacggtgacgtagaatcagatattgttgcacaaggatttggctccttggggttaatgacatctgtgttgattaccccggacggtaaaacctttgaaagcgaagccgcccacggtacagtaacaagacattttagacagcatcagcaaggaaaggagacgtcaacaaattccattgcatcaattttcgcgtggactagaggtattattcaaaggggtaaacttgataatactccagatgtagttaagttcggccaaatattggaaagcgctacggtaaatacagtgcaagaagatggaatcatgactaaagatttggcgctcattctcggtaagtctgaaagatccgcttatgtcactaccgaggagttcattgacgcggtggaatctagattgaaaaaagagttcgaggcagctgcattgtaa SEQ ID IDP2, S. cMTKIKVANPIVEMDGDEQTRIIWHLIRDKLVLPYLDVDLKYYDLSVEYR NO: 100 (Amino AcidDQTNDQVTVDSATATLKYGVAVKCATITPDEARVEEFHLKKMWKSPN Seq.)GTIRNILGGTVFREPIIIPRIPRLVPQWEKPIIIGRHAFGDQYKATDVIVPEEGELRLVYKSKSGTHDVDLKVFDYPEHGGVAMMMYNTTDSIEGFAKASFELAIERKLPLYSTTKNTILKKYDGKFKDVFEAMYARSYKEKFESLGIWYEHRLIDDMVAQMLKSKGGYIIAMKNYDGDVESDIVAQGFGSLGLMTSVLITPDGKTFESEAAHGTVTRHFRQHQQGKETSTNSIASIFAWTRGIIQRGKLDNTPDVVKFGQILESATVNTVQEDGIMTKDLALILGKSERSAYVTTEEFIDAVESRLKKEFEAAAL SEQ ID MAE1 (non-MWPIQQSRLYSSNTRSHKATTTRENTFQKPYSDEEVTKTPVGSRAR NO: 101 mitochondrial),KIFEAPHPHATRLTVEGAIECPLESFQLLNSPLFNKGSAFTQEEREAF Sc (AminoNLEALLPPQVNTLDEQLERSYKQLCYLKTPLAKNDFMTSLRVQNKVL Acid Seq.)YFALIRRHIKELVPIIYTPTEGDAIAAYSHRFRKPEGVFLDITEPDSIECRLATYGGDKDVDYIVVSDSEGILGIGDQGIGGVRIAISKLALMTLCGGIHPGRVLPVCLDVGTNNKKLARDELYMGNKFSRIRGKQYDDFLEKFIKAVKKVYPSAVLHFEDFGVKNARRLLEKYRYELPSFNDDIQGTGAVVMASLIAALKHTNRDLKDTRVLIYGAGSAGLGIADQIVNHMVTHGVDKEEARKKIFLMDRRGLILQSYEANSTPAQHVYAKSDAEWAGINTRSLHDVVENVKPTCLVGCSTQAGAFTQDVVEEMHKHNPRPIIFPLSNPTRLHEAVPADLMKWTNNNALVATGSPFPPVDGYRISENNNCYSFPGIGLGAVLSRATTITDKMISAAVDQLAELSPLREGDSRPGLLPGLDTITNTSARLATAVILQALEEGTARIEQEQVPGGAPGETVKVPRDFDECLQWVKAQMWE PVYRPMIKVQHDPSVHTNQLSEQ ID MAE1 (non- ATGTGGCCTATTCAGCAATCGCGTTTATATTCTTCTAACACTAGAT NO: 102mitochondrial), CGCATAAAGCTACCACAACAAGAGAAAATACTTTCCAAAAGCCATASc (Nucleic CAGCGACGAGGAGGTCACTAAAACACCCGTCGGTTCTCGCGCCA Acid Seq.)GAAAGATCTTCGAAGCTCCTCACCCACATGCCACTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGGAGAGCTTTCAACTTTTAAACTCTCCTTTATTTAACAAGGGTTCTGCATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGCATTGCTACCACCACAAGTGAACACTTTGGACGAACAACTGGAAAGAAGCTACAAGCAGTTATGCTATTTGAAGACGCCCTTGGCCAAAAACGACTTCATGACGTCTTTGAGAGTACAGAACAAAGTCCTATATTTTGCATTAATAAGGAGACATATCAAGGAATTAGTTCCTATCATTTACACCCCAACCGAAGGTGATGCTATTGCTGCCTATTCCCACAGGTTCAGAAAGCCAGAAGGTGTGTTTTTAGACATTACCGAACCTGATTCCATCGAATGTAGATTGGCTACATACGGTGGAGACAAAGATGTAGACTACATCGTTGTGTCGGATTCGGAAGGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTACGTATTGCTATCTCCAAATTGGCATTGATGACGCTGTGCGGTGGTATTCATCCCGGCCGTGTGCTACCTGTGTGTTTGGACGTCGGTACTAACAACAAGAAACTAGCCCGTGACGAATTGTACATGGGTAACAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTTCTTGGAAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAGCGCCGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCTAGAAGATTACTAGAAAAGTACAGGTACGAATTGCCATCATTCAACGATGACATTCAGGGCACCGGTGCCGTCGTGATGGCCTCGTTGATTGCTGCTTTGAAACATACCAACAGAGACTTGAAAGACACCAGAGTGCTTATTTACGGTGCCGGGTCTGCGGGCCTCGGTATCGCAGATCAAATTGTGAATCATATGGTCACGCACGGCGTTGACAAGGAAGAAGCGCGCAAGAAAATCTTCTTGATGGACAGACGTGGGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGCCCAACACGTATACGCTAAGAGTGATGCGGAATGGGCTGGTATCAACACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACCAACGTGTTTGGTTGGCTGCTCCACACAAGCAGGCGCATTCACTCAAGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGACCGATCATTTTCCCATTATCCAACCCTACTAGACTACACGAAGCCGTTCCTGCCGATTTAATGAAGTGGACCAACAACAACGCTCTTGTAGCTACCGGATCTCCTTTCCCACCTGTTGATGGTTACCGTATCTCGGAGAACAACAATTGTTACTCTTTCCCAGGTATCGGTTTAGGTGCCGTACTATCGCGTGCCACCACCATCACAGACAAGATGATCTCCGCTGCAGTGGACCAACTAGCCGAATTGTCGCCACTAAGAGAGGGCGACTCGAGACCTGGGTTGCTACCCGGCCTGGACACCATCACCAACACTTCTGCGCGTCTAGCTACCGCTGTGATCTTGCAAGCACTCGAGGAGGGAACCGCCCGTATCGAGCAAGAACAAGTACCGGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCCTCGTGACTTTGACGAATGTTTACAGTGGGTCAAAGCCCAAATGTGGGAGCCTGTGTACAGACCTATGATCAAGGTCCAACATGACCCATCGG TGCACACCAACCAATTGTAGSEQ ID MAE1, MLKFNKISARFVSSTATASATSGEMRTVKTPVGIKAAIESLKPKATRVS NO: 103Candida MDGPVECPLTDFALLNSPQFNKGSAFSLEERKSFKLTGLLPSQVNTL (Amino AcidDEQVERAYRQFTYLKTPLAKNDFCTSMRLQNKVLYYELVRRNIREML Seq.)PIIYTPTEGDAIASYSDRFRKPEGCFLDINDPDNIDERLAAYGENKDIDYIVMSDGEGIXXXSDRFRKPEGCFLDINDPDNIDERLAAYGENKDIDYIVMSDGEGILGIGDQGVGGIRIAIAKLGLMTLCGGIHPARVLPITLDVGTNNDRLLNDDLYMGNKFPRVRGERYWDFVDKVIHAITKRFPSAVMHYEDFGVTTGRDMLHKYRTALPSFNDDIQGTGAVVMASITAALKFSNRSLKDIEVLIYGAGSAGLGIADQITNHLVSHGATPEQARSRIHCMDRYGLITTESNNASPAQMNYADKASDWEGVDTSSLLACandidaEKVKPTVLVGCSTQAGAFTEEVVKTMYKYNPQPIIFPLSNPTRLHEAVPADLMKWTDNNALIATGSPFEPVDGYYISENNNCFTFPGIGLGAVLSRCSTISDTMISAAVDRLASMSPKMENPKNGLLPRLEEIDEVSAHVATAVILQSLKEGTARVESEKKPDGGYVEVPRDYDDCLKWVQSQMWKPVYRPYIKVEYVSNI HTYQY SEQ ID MAE1,atgctcaaattcaataaaatactggccagattcgtctcctccacggccaccgcatccgccacgtcaNO: 104 Candidaggggaaatgcgtaccgtcaagaccccagtggggatcaaggcggccatcgaatcattaaaacca(Nucleic AcidaaagctactagagtctccatggacggacctgtcgaatgcccattgaccgatttcgccttgttgaactcSeq.)ccctcaattcaacaaaggttcggcattttctttggaagaaaggaaaagtttcaagttgaccgggctcctcccttctcaagtcaacactttggatgaacaggttgaaagagcctatagacaattcacatacttgaagaccccattggccaagaacgatttctgcacgtctatgagattgcagaacaaagtgctttactacgagttggttagaagaaatatccgtgagatgttgcccatcatctacaccccaaccgaaggggacgccatcgccagttattccgacaggttcagaaaaccagagggctgtttcttggatatcaacgaccccgacaacatcgatgagagattagctgcctatggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcnnnnnnnnctccgacaggttcagaaaaccagagggctgcttcttggacatcaatgacccagacaacatcgacgagagattggctgcctatggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcctcggtattggagaccaaggcgtcggtggtatcagaattgccattgctaaattggggttgatgaccctttgtggtggtattcacccggccagagttttgcccatcactttggatgttggtacaaataacgacaggttgttgaatgatgatttgtacatgggcaacaagttccctagagtcagaggagaaagatactgggactttgtcgataaggtcatacacgcaattacgaaacggttcccaagtgccgtgatgcattacgaagatttcggagtcacaactggtagggacatgttgcacaagtaccgtacggctcttccttctttcaacgacgacatccaaggtaccggtgcagttgtcatggcatcgatcacagctgccttgaagttctccaaccgtagcctaaaggacatcgaggttttgatttacggtgccggctcagctggtttaggtattgctgaccagatcaccaaccacttggtcagccacggcgctactccagaacaagccagatctaggatccattgtatggaccgttatgggttgatcacaactgaatccaacaacgccagtcctgctcaaatgaactacgccgacaaggcatctgattgggaaggtgtcgatacctcgagtctacttgcctgtgttgagaaagtcaaaccaactgtcttggttgggtgttccactcaggcaggtgcattcaccgaagaggttgtcaaaaccatgtacaagtacaacccacagccaattattttcccattgtccaaccctaccagattgcatgaagccgtgccggctgatttgatgaaatggaccgacaacaacgcgttgattgccaccggttctccatttgaacctgtcgatggctactacatttccgaaaacaacaactgtttcaccttcccaggtattgggttgggtgctgtcttgtccagatgtagcaccatttcggataccatgatttctgccgccgttgatagattggcttcgatgtcgccaaagatggagaacccaaagaacggattgttgcctagattggaagaaatcgacgaagtcagtgcccatgttgccacggctgttatcttgcaatctttgaaggaaggcaccgctagagtcgaaagcgagaagaagccagacggtggttacgttgaagttccaagagactatgatgattgtcttaagtgggtgcaatcacaaatgtggaagccagtgtacagaccatacatcaaggttgagtacgtttcgaatattcacacctatcaatat SEQ ID MAE1, ScMLRTRLSVSVAARSQLTRSLTASRTAPLRRWPIQQSRLYSSNTRSHK NO: 105 (Amino AcidATTTRENTFQKPYSDEEVTKTPVGSRARKIFEAPHPHATRLTVEGAIE Seq.)CPLESFQLLNSPLFNKGSAFTQEEREAFNLEALLPPQVNTLDEQLERSYKQLCYLKTPLAKNDFMTSLRVQNKVLYFALIRRHIKELVPIIYTPTEGDAIAAYSHRFRKPEGVFLDITEPDSIECRLATYGGDKDVDYIVVSDSEGILGIGDQGIGGVRIAISKLALMTLCGGIHPGRVLPVCLDVGTNNKKLARDELYMGNKFSRIRGKQYDDFLEKFIKAVKKVYPSAVLHFEDFGVKNARRLLEKYRYELPSFNDDIQGTGAVVMASLIAALKHTNRDLKDTRVLIYGAGSAGLGIADQIVNHMVTHGVDKEEARKKIFLMDRRGLILQSYEANSTPAQHVYAKSDAEWAGINTRSLHDVVENVKPTCLVGCSTQAGAFTQDVVEEMHKHNPRPIIFPLSNPTRLHEAVPADLMKWTNNNALVATGSPFPPVDGYRISENNNCYSFPGIGLGAVLSRATTITDKMISAAVDQLAELSPLREGDSRPGLLPGLDTITNTSARLATAVILQALEEGTARIEQEQVPGGAPGETVKVPRDFDECLQWVKAQMWEPVYRPMIKVQHDPSVHTNQL SEQ ID MAE1, ScATGCTTAGAACCAGACTATCCGTTTCCGTTGCTGCTAGATCGCAAC NO: 106 (Nucleic AcidTAACCAGATCCTTGACAGCATCAAGGACAGCACCATTAAGAAGAT Seq.)GGCCTATTCAGCAATCGCGTTTATATTCTTCTAACACTAGATCGCATAAAGCTACCACAACAAGAGAAAATACTTTCCAAAAGCCATACAGCGACGAGGAGGTCACTAAAACACCCGTCGGTTCTCGCGCCAGAAAGATCTTCGAAGCTCCTCACCCACATGCCACTCGTTTGACTGTAGAAGGTGCCATAGAATGTCCCTTGGAGAGCTTTCAACTTTTAAACTCTCCTTTATTTAACAAGGGTTCTGCATTTACACAAGAAGAAAGGGAAGCGTTTAATTTAGAAGCATTGCTACCACCACAAGTGAACACTTTGGACGAACAACTGGAAAGAAGCTACAAGCAGTTATGCTATTTGAAGACGCCCTTGGCCAAAAACGACTTCATGACGTCTTTGAGAGTACAGAACAAAGTCCTATATTTTGCATTAATAAGGAGACATATCAAGGAATTAGTTCCTATCATTTACACCCCAACCGAAGGTGATGCTATTGCTGCCTATTCCCACAGGTTCAGAAAGCCAGAAGGTGTGTTTTTAGACATTACCGAACCTGATTCCATCGAATGTAGATTGGCTACATACGGTGGAGACAAAGATGTAGACTACATCGTTGTGTCGGATTCGGAAGGTATTCTGGGAATTGGTGACCAAGGTATCGGTGGTGTACGTATTGCTATCTCCAAATTGGCATTGATGACGCTGTGCGGTGGTATTCATCCCGGCCGTGTGCTACCTGTGTGTTTGGACGTCGGTACTAACAACAAGAAACTAGCCCGTGACGAATTGTACATGGGTAACAAGTTCTCCAGAATCAGGGGTAAGCAATATGACGACTTCTTGGAAAAATTCATCAAGGCCGTTAAGAAAGTGTATCCAAGCGCCGTTCTGCATTTCGAAGATTTCGGTGTTAAGAACGCTAGAAGATTACTAGAAAAGTACAGGTACGAATTGCCATCATTCAACGATGACATTCAGGGCACCGGTGCCGTCGTGATGGCCTCGTTGATTGCTGCTTTGAAACATACCAACAGAGACTTGAAAGACACCAGAGTGCTTATTTACGGTGCCGGGTCTGCGGGCCTCGGTATCGCAGATCAAATTGTGAATCATATGGTCACGCACGGCGTTGACAAGGAAGAAGCGCGCAAGAAAATCTTCTTGATGGACAGACGTGGGTTAATTCTACAATCTTACGAGGCTAACTCCACTCCCGCCCAACACGTATACGCTAAGAGTGATGCGGAATGGGCTGGTATCAACACCCGCTCTTTACATGATGTGGTGGAGAACGTCAAACCAACGTGTTTGGTTGGCTGCTCCACACAAGCAGGCGCATTCACTCAAGATGTCGTAGAAGAAATGCACAAGCACAATCCTAGACCGATCATTTTCCCATTATCCAACCCTACTAGACTACACGAAGCCGTTCCTGCCGATTTAATGAAGTGGACCAACAACAACGCTCTTGTAGCTACCGGATCTCCTTTCCCACCTGTTGATGGTTACCGTATCTCGGAGAACAACAATTGTTACTCTTTCCCAGGTATCGGTTTAGGTGCCGTACTATCGCGTGCCACCACCATCACAGACAAGATGATCTCCGCTGCAGTGGACCAACTAGCCGAATTGTCGCCACTAAGAGAGGGCGACTCGAGACCTGGGTTGCTACCCGGCCTGGACACCATCACCAACACTTCTGCGCGTCTAGCTACCGCTGTGATCTTGCAAGCACTCGAGGAGGGAACCGCCCGTATCGAGCAAGAACAAGTACCGGGAGGAGCTCCCGGCGAAACTGTCAAGGTTCCTCGTGACTTTGACGAATGTTTACAGTGGGTCAAAGCCCAAATGTGGGAGCCTGTGTACAGACCTATGATCAAGGTCCAACATGACCCATCGGTGC ACACCAACCAATTGTAG SEQ IDPYC2, Sc MSSSKKLAGLRDNFSLLGEKNKILVANRGEIPIRIFRSAHELSMRTIAIY NO: 107(Amino Acid SHEDRLSMHRLKADEAYVIGEEGQYTPVGAYLAMDEIIEIAKKHKVDFI Seq.)HPGYGFLSENSEFADKVVKAGITWIGPPAEVIDSVGDKVSARHLAARANVPTVPGTPGPIETVQEALDFVNEYGYPVIIKAAFGGGGRGMRVVREGDDVADAFQRATSEARTAFGNGTCFVERFLDKPKHIEVQLLADNHGNVVHLFERDCSVQRRHQKVVEVAPAKTLPREVRDAILTDAVKLAKVCGYRNAGTAEFLVDNQNRHYFIEINPRIQVEHTITEEITGIDIVSAQIQIAAGATLTQLGLLQDKITTRGFSIQCRITTEDPSKNFQPDTGRLEVYRSAGGNGVRLDGGNAYAGATISPHYDSMLVKCSCSGSTYEIVRRKMIRALIEFRIRGVKTNIPFLLTLLTNPVFIEGTYWTTFIDDTPOLFQMVSSONRAQKLLHYLADLAVNGSSIKGQIGLPKLKSNPSVPHLHDAQGNVINVTKSAPPSGWRQVLLEKGPSEFAKQVRQFNGTLLMDTTWRDAHQSLLATRVRTHDLATIAPTTAHALAGAFALECWGGATFDVAMRFLHEDPWERLRKLRSLVPNIPFQMLLRGANGVAYSSLPDNAIDHFVKQAKDNGVDIFRVFDALNDLEQLKVGVNAVKKAGGVVEATVCYSGDMLQPGKKYNLDYYLEVVEKIVQMGTHILGIKDMAGTMKPAAAKLLIGSLRTRYPDLPIHVHSHDSAGTAVASMTACALAGADVVDVAINSMSGLTSQPSINALLASLEGNIDTGINVEHVRELDAYWAEMRLLYSCFEADLKGPDPEVYQHEIPGGQLTNLLFQAQQLGLGEQWAETKRAYREANYLLGDIVKVTPTSKVVGDLAQFMVSNKLTSDDIRRLANSLDFPDSVMDFFEGLIGQPYGGFPEPLRSDVLRNKRRKLTCRPGLELEPFDLEKIREDLQNRFGDIDECDVASYNMYPRVYEDFQKIRETYGDLSVLPTKNFLAPAEPDEEIEVTIEQGKTLIIKLQAVGDLNKKTGQREVYFELNGELRKIRVADKSQNIQSVAKPKADVHDTHQIGAPMAGVIIEVKVHKGSLVKKGESIAVLSAMKMEMVVSSPADGQVKDVFIKDGESVDASDLLVVLEEETLPPSQKK* SEQ ID PYC2, ScATGAGCAGTAGCAAGAAATTGGCCGGTCTTAGGGACAATTTCAGT NO: 108 (Nucleic AcidTTGCTCGGCGAAAAGAATAAGATCTTGGTCGCCAATAGAGGTGAA Seq.)ATTCCGATTAGAATTTTTAGATCTGCTCATGAGCTGTCTATGAGAACCATCGCCATATACTCCCATGAGGACCGTCTTTCAATGCACAGGTTGAAGGCGGACGAAGCGTATGTTATCGGGGAGGAGGGCCAGTATACACCTGTGGGTGCTTACTTGGCAATGGACGAGATCATCGAAATTGCAAAGAAGCATAAGGTGGATTTCATCCATCCAGGTTATGGGTTCTTGTCTGAAAATTCGGAATTTGCCGACAAAGTAGTGAAGGCCGGTATCACTTGGATCGGCCCTCCAGCTGAAGTTATTGACTCTGTGGGTGACAAAGTCTCTGCCAGACACTTGGCAGCAAGAGCTAACGTTCCTACCGTTCCCGGTACTCCAGGACCTATCGAAACTGTGCAAGAGGCACTTGACTTCGTTAATGAATACGGCTACCCGGTGATCATTAAGGCCGCCTTTGGTGGTGGTGGTAGAGGTATGAGAGTCGTTAGAGAAGGTGACGACGTGGCAGATGCCTTTCAACGTGCTACCTCCGAAGCCCGTACTGCCTTCGGTAATGGTACCTGCTTTGTGGAAAGATTCTTGGACAAGCCAAAGCATATTGAAGTTCAATTGTTGGCTGATAACCACGGAAACGTGGTTCATCTTTTCGAAAGAGACTGTTCTGTGCAAAGAAGACACCAAAAAGTTGTCGAAGTCGCTCCAGCAAAGACTTTGCCCCGTGAAGTTCGTGACGCTATTTTGACAGATGCTGTTAAATTAGCTAAGGTATGTGGTTACAGAAACGCAGGTACCGCCGAATTCTTGGTTGACAACCAAAACAGACACTATTTCATTGAAATTAATCCAAGAATTCAAGTGGAGCATACCATCACTGAAGAAATCACCGGTATTGACATTGTTTCTGCCCAAATCCAGATTGCCGCAGGTGCCACTTTGACTCAACTAGGTCTATTACAGGATAAAATCACCACCCGTGGGTTTTCCATCCAATGTCGTATTACCACTGAAGATCCCTCTAAGAATTTCCAACCGGATACCGGTCGCCTGGAGGTCTATCGTTCTGCCGGTGGTAATGGTGTGAGATTGGACGGTGGTAACGCTTATGCAGGTGCTACTATCTCGCCTCACTACGACTCAATGCTGGTCAAATGTTCATGCTCTGGTTCTACTTATGAAATCGTCCGTAGGAAGATGATTCGTGCCCTGATCGAATTCAGAATCAGAGGTGTTAAGACCAACATTCCCTTCCTATTGACTCTTTTGACCAATCCAGTTTTTATTGAGGGTACATACTGGACGACTTTTATTGACGACACCCCACAACTGTTCCAAATGGTATCGTCACAAAACAGAGCGCAAAAACTGTTACACTATTTGGCAGACTTGGCAGTTAACGGTTCTTCTATTAAGGGTCAAATTGGCTTGCCAAAACTAAAATCAAATCCAAGTGTCCCCCATTTGCACGATGCTCAGGGCAATGTCATCAACGTTACAAAGTCTGCACCACCATCCGGATGGAGACAAGTGCTACTGGAAAAGGGACCATCTGAATTTGCCAAGCAAGTCAGACAGTTCAATGGTACTCTACTGATGGACACCACCTGGAGAGACGCTCATCAATCTCTACTTGCAACAAGAGTCAGAACCCACGATTTGGCTACAATCGCTCCAACAACCGCACATGCCCTTGCAGGTGCTTTCGCTTTAGAATGTTGGGGTGGTGCTACATTCGACGTTGCAATGAGATTCTTGCATGAGGATCCATGGGAACGTCTGAGAAAATTAAGATCTCTGGTGCCTAATATTCCATTCCAAATGTTATTACGTGGTGCCAACGGTGTGGCTTACTCTTCATTACCTGACAATGCTATTGACCATTTTGTCAAGCAAGCCAAGGATAATGGTGTTGATATATTTAGAGTTTTTGATGCCTTGAATGATTTAGAACAATTAAAAGTTGGTGTGAATGCTGTCAAGAAGGCCGGTGGTGTTGTCGAAGCTACTGTTTGTTACTCTGGTGACATGCTTCAGCCAGGTAAGAAATACAACTTAGACTACTACCTAGAAGTTGTTGAAAAAATAGTTCAAATGGGTACACATATCTTGGGTATTAAGGATATGGCAGGTACTATGAAACCGGCCGCTGCCAAATTATTAATTGGCTCCCTAAGAACCAGATATCCGGATTTACCAATTCATGTTCACAGTCATGACTCCGCAGGTACTGCTGTTGCGTCTATGACTGCATGTGCCCTAGCAGGTGCTGATGTTGTCGATGTAGCTATCAATTCAATGTCGGGCTTAACTTCCCAACCATCAATTAATGCACTGTTGGCTTCATTAGAAGGTAACATTGATACTGGGATTAACGTTGAGCATGTTCGTGAATTAGATGCATACTGGGCCGAAATGAGACTGTTGTATTCTTGTTTCGAGGCCGACTTGAAGGGACCAGATCCAGAAGTTTACCAACATGAAATCCCAGGTGGTCAATTGACTAACTTGTTATTCCAAGCTCAACAACTGGGTCTTGGTGAACAATGGGCTGAAACTAAAAGAGCTTACAGAGAAGCCAATTACCTACTGGGAGATATTGTTAAAGTTACCCCAACTTCTAAGGTTGTCGGTGATTTAGCTCAATTCATGGTTTCTAACAAACTGACTTCCGACGATATTAGACGTTTAGCTAATTCTTTGGACTTTCCTGACTCTGTTATGGACTTTTTTGAAGGTTTAATTGGTCAACCATACGGTGGGTTCCCAGAACCATTAAGATCTGATGTATTGAGAAACAAGAGAAGAAAGTTGACGTGCCGTCCAGGTTTAGAATTAGAACCATTTGATCTCGAAAAAATTAGAGAAGACTTGCAGAACAGATTCGGTGATATTGATGAATGCGATGTTGCTTCTTACAATATGTATCCAAGGGTCTATGAAGATTTCCAAAAGATCAGAGAAACATACGGTGATTTATCAGTTCTACCAACCAAAAATTTCCTAGCACCAGCAGAACCTGATGAAGAAATCGAAGTCACCATCGAACAAGGTAAGACTTTGATTATCAAATTGCAAGCTGTTGGTGACTTAAATAAGAAAACTGGGCAAAGAGAAGTGTATTTTGAATTGAACGGTGAATTAAGAAAGATCAGAGTTGCAGACAAGTCACAAAACATACAATCTGTTGCTAAACCAAAGGCTGATGTCCACGATACTCACCAAATCGGTGCACCAATGGCTGGTGTTATCATAGAAGTTAAAGTACATAAAGGGTCTTTGGTGAAAAAGGGCGAATCGATTGCTGTTTTGAGTGCCATGAAAATGGAAATGGTTGTCTCTTCACCAGCAGATGGTCAAGTTAAAGACGTTTTCATTAAGGATGGTGAAAGTGTTGACGCATCAGATTTGTTGGTTGTCCTAGAAGAAGAAACCCTACCCCCATCCCAAAAAAAGTAA SEQ ID GUT2,MSRFLKSTFAKSALAAGAAVGGTIVYLDFIKPKNETHLATTYPAFNKNI NO: 109 CandidaPAPPPRESLIENLKKTPQFDVLVIGGGAVGTGTALDAATRGLNVCLLE (Amino AcidKTDFGAGTSSKSTKMAHGGVRYLEKAIFQLSRAQLDLVIEALNERGN Seq.)MLRTAPHLCSVLPIMIPVYNWWQVPYFFAGCKMYDWFAGKQNLRSSTIFTTEQAAAIAPMMDTSNLKAACandidaYHDGSFNDTRYNVSLAVTAIKNGATVLNYFEVEQLLKDDKGKLYGVKAKDLETNETYEIKATSVVNATGPFADKILEMDEDPKGLPPKVEQPPRMVVPSSGVHIVLPEYYCPTNYGLLDPSTSDGRVMFFLPWQGKVLAGTTDTPLKTVPANPVPTEEEIQDIIKEMQKYLVFPIDRNDVLSAWSGIRPLVRDPSTIPKGQEGSGKTEGLVRSHLLVQSPTGLVTISGGKWTTYREMAQETVDYLVDHFSYGDKKLLPCQTNKLLLVGGENYTKNYSARLIHEYKIPLKLAKHLSHNYGSRAPLVLDLYAESDFNKLPVTLAATKEFEPSEKKANEDNQLSYQSFDEPFTVAELKYSLKYEYPRTPLDFLARRTRLAFLNAREALNAVDGVVEIMSQEYGWDKETEDRLRKEARQYIGNMGISPKKFDVEKIVIQ SEQ ID GUT2,atgtcaagattcttaaagtcaacctttgcaaagtcagccttagctgctggtgctgcagtcggtggtacNO: 110 Candidacatcgtgtacttggatttcatcaagccaaagaacgaaacccacttggccactacctacccagcctt(Nucleic Acidcaacaagaacataccagctcctcccccacgtgagtccttgatcgaaaacttgaagaagactcctc Seq.)aattcgacgtcttggtcattggtggtggtgcagttggtaccggtactgctctcgacgctgccacgcgtgggttaaatgtgtgtcttttggaaaaaaccgacttcggtgcaggaacttcgtcgaagtccaccaaaatggcccacggtggtgtccgttacttggagaaggccatttttcaattgtccagagcccaattggacttggtcattgaagccttgaacgaaagaggtaacatgttgagaactgctcctcacttgtgctctgttttgccaatcatgattccagtctacaactggtggcaggttccttactttttcgctggttgtaagatgtacgattggtttgccggtaagcaaaacttgcgttcctccactatcttcaccactgaacaggctgccgctattgccccaatgatggatacttctaacttgaaagccgcctgtgtctaccacgatggtagtttcaacgataccagatacaacgtctccttggctgtcaccgccatcaagaacggcgccactgtcttgaactatttcgaagttgagcaattgttgaaagatgacaagggtaagttgtacggtgtcaaggccaaggatttggaaaccaacgagacctacgagatcaaagccactagtgttgtcaacgctaccggtcctttcgctgataagatcttggagatggacgaagatccaaagggtttgcctccaaaggtcgagcaaccaccaagaatggttgttccatcttccggtgtccacattgttttgcctgaatactactgcccaaccaactacggtttgttagacccatccacctccgacggtagagtcatgttctttttgccatggcaaggtaaggtcttggctggtaccactgatactccattgaagactgttcctgccaaccctgttccaactgaggaagaaatccaagacatcatcaaagaaatgcaaaagtaccttgttttcccaatcgacagaaacgacgttttgtctgcttggtccggtatcagaccattggttagagacccatctactattccaaagggccaagaaggctctggtaagactgaaggtttagttcgttcccacttgttagttcaatccccaactggtttggtcaccatctccggtggtaaatggaccacctacagagaaatggcccaagaaactgttgactacctcgttgatcactttagctacggcgacaagaagctcttgccatgccagaccaacaaattgctcttggttggtggtgaaaactacaccaagaactactctgccagattgatccacgaatacaagatcccattgaagttggccaagcacttgtcccacaactacggttccagagctccattggtcttggacttgtacgctgaaagtgatttcaacaagttgccagtcaccttggctgccaccaaggagtttgagccatctgaaaagaaggccaacgaagacaaccagttgagttaccagagtttcgacgagccattcaccgttgctgagttgaagtactccttgaagtacgagtacccaagaaccccactcgatttcttggccagaagaacgagattggctttcttgaacgccagagaagccctcaatgcggttgatggggttgttgaaatcatgagtcaagagtacggctgggacaaggagaccgaagatagattgagaaaagaagccagacaatacattggtaatatgggtatttctccaaagaaatttgacgttgaaaagattgttattcaa SEQ ID gpsA, AfMIVSILGAGAMGSALSVPLVDNGNEVRIWGTEFDTEILKSISAGREHP NO: 111 (Amino AcidRLGVKLNGVEIFWPEQLEKCLENAEVVLLGVSTDGVLPVMSRILPYLK Seq.)DQYIVLISKGLIDFDNSVLTVPEAVWRLKHDLRERTVAITGPAIAREVAKRMPTTVVFSSPSESSANKMKEIFETEYFGVEVTTDIIGTEITSALKNVYSIAIAWIRGYESRKNVEMSNAKGVIATRAINEMAELIEILGGDRETAFGLSGFGDLIATFRGGRNGMLGELLGKGLSIDEAMEELERRGVGVVEGYKTAEKAYRLSSKINADTKLLDSIYRVLYEGLKVEEVLFELATFK SEQ ID gpsA, AfatgattgtttcgatactgggagcgggtgcaatgggctcagccctctccgtcccgctcgtagataacgNO: 112 (Nucleic Acidgcaacgaagtgagaatctgggggaccgagttcgatacggagattttaaaatcaatctcagccgg Seq.)cagagagcatccaaggcttggtgtaaagctcaatggcgtggaaattttctggccagagcagcttgaaaaatgtttggagaatgcagaggttgtacttctgggtgttagcacggatggcgtgctgcccgtaatgagcagaattctcccgtatctcaaggaccagtacatcgtactcatctctaaagggctgattgattttgataacagtgttctgacggttcccgaagctgtatggaggttaaagcacgatttgagggaaaggactgtggcgataaccgggcccgctattgcaagagaggtggcgaaacgcatgcccacaaccgttgttttcagcagcccatccgaaagctcggccaataaaatgaaagaaatctttgagacagagtactttggcgttgaagtaacaacagacataattggcacggaaataacctccgccctcaaaaacgtttattccatagccattgcatggataaggggctacgagagcagaaaaaacgttgagatgagcaatgcaaagggagtgattgcaacgagagccataaacgagatggcagagctgatagagattctcggaggggatagagagaccgcctttggcctttccggatttggagacctcatcgcaaccttcaggggaggaaggaacgggatgctgggagagctgcttggaaaggggcttagcatcgatgaggcgatggaggagcttgagaggagaggagttggtgtggttgagggctacaaaacggcagagaaagcatacaggctgtccagcaaaataaatgcagacacaaagctgctcgacagcatctacagagtcctttatgaaggactgaaggttgaggaagtgctgtttgaactcgctacatttaaataa SEQ ID ADH1-2,MHALFSKSVFLKYVSSPTTSAIPHSLEFIVSRSSYLRRRIPPYLPRCSH NO: 113 CandidaFPSFYYSSSSVYTKKSFHTMSANIPKTQKAVVFEKNGGELKYKDIPVP (Amino AcidTPKANELLINVKYSGVCHTDLHAWKGDWPLDTKLPLVGGHEGAGVV Seq.)VGMGENVKGWKIGDFAGIKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGSFEQYATADAVQAARIPAGTDLAEVAPILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGDFVKSLGAEAYIDFLKEKGIVAAVKKATDGGPHGAINVSVSEKAIDQSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGLSELPQVFKLMEEGKILGRYVLDTSK SEQ ID ADH1-2,atgtctgctaatatcccaaaaactcaaaaagctgtcgtcttcgagaagaacggtggtgaattaaaaNO: 114 Candidatacaaagacatcccagtgccaaccccaaaggccaacgaattgctcatcaacgtcaagtactcgg(Nucleic AcidgtgtctgtcacactgatttgcacgcctggaagggtgactggccattggacaccaaattgccattggttSeq.) ggtggtcacgaaggtgctggtgttgttgtcggcatgggtgaaaacgtcaagggctggaaaatcggtgatttcgccggtatcaaatggttgaacggttcttgtatgtcctgtgagttctgtcagcaaggtgctgaaccaaactgtggtgaagctgacttgtctggttacacccacgatggttctttcgaacaatacgccactgctgatgctgtgcaagccgccagaatcccagctggcactgatttggccgaagttgccccaatcttgtgtgctggtgtcaccgtctacaaagccttgaagactgccgacttggctgctggtcaatgggtcgctatctccggtgctggtggtggtttgggctccttggctgtccaatacgccgtcgccatgggtttgagagtcgttgccattgacggtggtgacgaaaagggtgactttgtcaagtccttgggtgctgaagcctacattgatttcctcaaggaaaagggcattgttgctgctgtcaagaaggccactgatggcggtccacacggtgctatcaatgtttccgtttccgaaaaagccattgaccaatctgtcgagtacgttagaccattgggtaaggttgttttggttggtttgccagctggctccaaggtcactgctggtgttttcgaagccgttgtcaagtccattgaaatcaagggttcttacgtcggtaacagaaaggatactgccgaagccgttgactttttctccagaggcttgatcaagtgtccaatcaagattgtgggcttgagtgaattgccacaggtcttcaagttgatggaagaaggtaagatcttgggtagatacgtcttggatacctccaaa SEQ ID ADH2a,MSIPTTQKAIIFETNGGKLEYKDIPVPKPKPNELLINVKYSGVCHTDLHA NO: 115 CandidaWKGDWPLDTKLPLVGGHEGAGVVVAIGDNVKGWKVGDLAGVKWLN (Amino AcidGSCMNCEYCQQGAEPNCPQADLSGYTHDGSFQQYATADAVQAARI Seq.)PAGTDLANVAPILCAGVTVYKALKTADLQPGQWVAISGAAGGLGSLAVQYAKAMGYRVVAIDGGADKGEFVKSLGAEVFVDFLKEKDIVGAVKKATDGGPHGAVNVSISEKAINQSVDYVRTLGKVVLVGLPAGSKVSAPVFDSVVKSIQIKGSYVGNRKDTAEAVDFFSRGLIKCPIKVVGLSELPEVY KLMEEGKILGRYVLDNSKSEQ ID ADH2a,atgtcaattccaactactcaaaaagctatcattttcgaaaccaacggtggaaaattagaatacaagNO: 116 Candidagacatcccagttccaaagccaaagccaaacgaattgctcatcaacgtcaagtactccggtgtctg(Nucleic Acidccacactgatttacacgcctggaagggtgactggccattggacaccaagttgccattggtgggtgg Seq.)tcacgaaggtgctggtgttgttgttgccattggtgacaatgtcaagggatggaaggtcggtgatttggccggtgtcaagtggttgaacggttcctgtatgaactgtgagtactgtcaacagggtgccgaaccaaactgtccacaggctgacttgtctggttacacccacgacggttctttccagcaatacgccactgcagatgccgtgcaagccgctagaattccagctggtactgatttagccaacgttgcccccatcttgtgtgctggtgtcactgtttacaaggccttgaagaccgccgacttgcagccaggtcaatgggtcgccatttccggtgccgctggtggtttgggttctttggccgttcaatacgccaaggccatgggctacagagttgtcgccatcgatggtggtgccgacaagggtgagttcgtcaagtctttgggcgctgaggtctttgttgatttcctcaaggaaaaggacattgttggtgctgtcaagaaggcaaccgatggtggcccacacggtgccgttaacgtttccatctccgaaaaggccatcaaccaatctgtcgactacgttagaaccttgggtaaggttgtcttggtcggtttgccagctggctccaaggtttctgctccagtctttgactccgtcgtcaagtccatccaaatcaagggttcctatgtcggtaacagaaaggacactgccgaagctgttgactttttctccagaggcttgatcaagtgtccaatcaaggttgtcggtttgagtgaattgccagaagtctacaagttgatggaagaaggtaagatcttgggtagatacgtcttggacaactctaag SEQ ID ADH2b,MSIPTTQKAVIYEANSAPLQYTDIPVPVPKPNELLVHVKYSGVCHSDIH NO: 117 CandidaVWKGDWFPASKLPVVGGHEGAGVVVAIGENVQGWKVGDLAGIKML (Aminio AcidNGSCMNCEYCQQGAEPNCPHADVSGYSHDGTFQQYATADAVQAAK Seq.)FPAGSDLASIAPISCAGVTVYKALKTAGLQPGQWVAISGAAGGLGSLAVQYAKAMGLRVVAIDGGDERGVFVKSLGAEVFVDFTKEANVSEAIIKATDGGAHGVINVSISEKAINQSVEYVRTLGTVVLVGLPAGAKLEAPIFNAVAKSIQIKGSYVGNRRDTAEAVDFFARGLVKCPIKVVGLSELPEIFKLL EEGKILGRYVVDTAK SEQ IDADH2b,atgtcaattccaactacccaaaaagctgttatctacgaagccaactctgctccattgcaatacaccgNO: 118 Candidaatatcccagttccagtccctaagccaaacgaattgctcgtccacgtcaaatactccggtgtttgtcac(Nucleic AcidtcagatatacacgtctggaagggtgactggttcccagcatcgaaattgcccgttgttggtggtcacgSeq.) aaggtgccggtgttgtcgttgccattggtgaaaacgtccaaggctggaaagtaggtgacttggcaggtataaagatgttgaatggttcctgtatgaactgtgaatactgtcaacaaggtgctgaaccaaactgtccccacgctgatgtctcgggttactcccacgacggtactttccaacagtacgctaccgccgatgctgttcaagctgctaaattcccagctggttctgatttagctagcatcgcacctatatcctgcgccggtgttactgtttacaaagcattgaaaactgcaggcttgcagccaggtcaatgggttgccatctctggtgcagctggtggtttgggttctttggctgtgcaatacgccaaggccatgggtttgagagtcgtggccattgacggtggtgacgaaagaggagtgtttgtcaaatcgttgggtgctgaagttttcgttgatttcaccaaagaggccaatgtctctgaggctatcatcaaggctaccgacggtggtgcccatggcgtcatcaacgtttccatttctgaaaaagccatcaaccagtctgttgaatatgttagaactttgggaactgttgtcttggttggtttgccagctggtgcaaagctcgaagctcctatcttcaatgccgttgccaaatccatccaaatcaaaggttcttacgtgggaaacagaagagacactgctgaggctgttgatttcttcgctagaggtttggtcaaatgtccaattaaggttgttgggttgagtgaattgccagagattttcaaattgttggaagagggtaagatcttgggtagatacgttgttgacactgccaag SEQ ID ADH3 (AminoMSTQSGYGYVKGQKTIQKYTDIPIPTPGPNEVLLKVEAAGLCLSDPHT NO: 119 Acid Seq.)LIGGPIESKPPLPNATKFIMGHEIAGSISQVGANLANDPYYKKGGRFALTIAQACGICENCRDGYDAKCESTTQAYGLNEDGGFQQYLLIKNLRTMLPIPEGVSYEEAAVSTDSVLTPFHAIQKVAHLLHPTTKVLVQGCGGLGFNAIQILKSYNCYIVATDVKPELEKLALEYGANEYHTDLTKSKHEPMSFDLIFDLVGIQPTFDLSDRYIKARGKILMIGLGRSKLFIPNYKLGIREVEIIFNFGGTSAEQIECMKWVAKGLIKPNIHVADFASLPEYLEDLAKGKLTGR IVFRPSKL SEQ ID ADH3Atgtcaactcaatcaggttacggatacgtgaaaggacaaaagaccattcagaaatacaccgac NO: 120(Nucleic AcidatcccgatccctacgccgggccccaacgaagtcttgttgaaagtcgaagctgccggcttgtgtctctSeq.) cggatccacacacgttgatcgggggtcccattgagagcaagccgccgttgccgaacgccacgaagttcatcatgggtcacgaaatcgcggggctgattagccaagtaggcgccaacttggccaacgatccatactataaaaagggaggtaggttcgccttgactatcgcgcaggcttgtgggatttgtgagaattgtcgtgatgggtatgatgcaaagtgtgagtctacgacgcaggcttatgggttgaacgaggacggtggattccagcaatacttgttgattaagaacttgcgtacgatgttgcctatccctgagggtgtgagttacgaagaagccgctgtgtctactgactctgtgttgactccattccatgcgattcagaaggtcgctcatttgttgcacccaactactaaggtgttggttcagggttgtggtgggttaggcttcaacgctattcaaatattgaagagctacaattgttacattgttgccactgatgtcaaaccagagcttgaaaaattagctttggagtatggtgccaacgaataccacactgatctcaccaagtccaagcatgagccaatgtcgttcgatttgattttcgaccttgtgggaatccaacctacttttgatttgtccgacaggtacatcaaagcaaggggtaagattcttatgattggcttaggcagatccaagttgtttattccaaattataaattgggtatccgtgaagtcgagatcattttcaattttggtggtacttcggccgagcaaattgagtgcatgaaatgggttgcaaaaggcttgatcaaacctaatattcacgtggctgattttgcttccttgcctgagtacctcgaggacttggccaagggtaaactcactggtagaattgtatttagaccaagtaagttg SEQ ID ADH4 (AminoMSLSGKTSLIAAGTKNLGGASAKELAKAGSNLFLHYRSNPDEAEKFK NO: 121 Acid Seq.)QEILKEFPNVKVETYQSKLDRAADLTNLFAAAKKAFPSGIDVAVNFVGKVIKGPITEVTEEQFDEMDVANNKIAFFFIKEAAINLNKNGSIISIVTSLLPAYTDSYGLYQGTKGAVEYYSKSISKELIPKGITSNCIGPGPASTSFLFNSETKESVEFFKTVAIDQRLTEDSDIAPIVLFLATGGRWATGQTIYASGG FTAR SEQ ID ADH4AtgtcattatcaggaaagacctcattaattgctgctggtaccaagaacttgggtggtgcaagtgccaNO: 122 (Nucleic Acidaagaattggccaaagccggctccaacctcttcttgcactacagatccaacccagacgaggctga Seq.)aaagttcaagcaagagatcctcaaggagttccctaacgtcaaggtcgaaacctaccaatccaaattggaccgtgccgccgacctcaccaacttgtttgctgctgccaagaaggcattccctagtggtattgacgtcgctgtcaactttgtcggtaaggtcatcaagggcccaatcactgaggtcactgaagaacagtttgacgagatggatgttgccaacaacaagattgcctttttcttcatcaaggaggccgctatcaacttgaacaagaacggtagtatcatttccatcgttactagtttgctcccagcttacaccgattcttacggtttgtaccagggtactaaaggagctgttgaatactattcgaaatctatcctgaaggagttgattccaaagggtatcaccagtaactgtattggtcctggtcctgcttctacttcctttttgtttaattccgaaaccaaggagagtgttgagttcttcaagaccgttgctattgaccaacgtttgactgaagacagcgacattgccccaattgtgttgttcctcgccactggaggtcgttgggcaactggtcaaactatttacgctagtggtggtttcactgctcgt SEQ ID ADH5 (Amino MSLVLKRLLPIRSPTLLNSKFIQLQSQIRTMAIPATQTGFFFTKQEGLNNO: 123 Acid Seq.) YRTDIPVRKPQAGQLLLKVNAVGLCHSDLHVIDKELECGDNYVMGHEIAGTVAEVGPEVEGYKVGDRVACandidaGPNGCGVCKHCLTGNDNVCKTAFLDWFGLGSDGGYEEYLLVRRPRNLVKVPDNVSIEEAAAITDAVLTPYHAVKTAKVKPTSNVLVIGAGGLGGNGIQIVKAFGGKVTVVDKKDKARDQAKALGADEVYSEIPASIEPGTFDVCLDFVSVQATYDLCQKYCEPKGIIIPVGLGATKLTIDLADLDLREITVTGTFWGTANDLREAFDLVSQGKIKPIVSHAPLKELPNYMEKLKQGAYEGRVVFHP SEQ ID ADH5AtgtcacttgtcctcaagcgattacttccaatcagatctcctactttactcaattcgaagttcatacagttNO: 124 (Nucleic AcidacaatctcaaattcgcacaatggctatccccgctactcaaactggattcttcttcaccaaacaagaaSeq.) ggtttaaactacagaaccgacattcctgtccgcaagccacaagccggtcagttgttgttgaaggtcaatgccgttggtctctgccactcggacttgcacgtgattgacaaggagcttgaatgtggtgacaactatgtcatgggccacgaaattgccggtaccgttgctgaagttggtcccgaagttgaaggctacaaggttggcgaccgtgtcgcttgtgttggtcctaacgggtgcggtgtctgtaagcactgcttgactggtaacgacaatgtctgtaagactgctttcctcgactggttcgggttgggctccgatggtgggtacgaagagtacttgttggtgagaagaccaagaaacttggttaaggtcccggacaacgtctcgattgaggaggctgctgctatcactgatgctgtgttgactccttaccatgctgtcaagactgccaaggtcaagccaaccagtaacgttttggttattggtgctggtggattaggtggtaacggtatccagattgtcaaggcttttggcggtaaggttactgttgtcgataagaaggataaggcacgtgaccaagctaaggctttgggtgctgatgaagtctacagtgaaatcccagcaagtattgaaccgggtacttttgatgtctgtcttgattttgtttccgtgcaagccacctatgatctctgccaaaagtactgtgagccaaagggtatcattatcccagttgggttgggtgctaccaagctcaccattgatttggcagatttggatctccgtgaaatcacggttactggtaccttctggggaactgccaatgacttgagagaggcgtttgatttggttagtcaaggtaagatcaagccgattgtttcacatgccccattgaaggagttgccaaactatatggagaagttgaagcagggagcatatgaaggaagagttgtcttccaccca SEQ ID ADH7 (AminoMTVDASSVPDKFQGFASDKRENWEHPKLISYDRKQLNDHDVVLKNE NO: 125 Acid Seq.)TCGLCYSDIHTLRSTWGPYGTNELVVGHEICGTVIAVGPKVTEFKVGDRAGIGAASSSCRHCSRCTHDNEQYCKEQVSTYNSVDPKAAGYVTKGGYSSHSIADELFVFKVPDDLPFEYASPLFCAGITTFSPLYRNLVGSDKDATGKTVGIIGVGGLGHLAIQFASKALNAKVVAFSRSSSKKEEALELGAAEFVATNEDKNWTSRYEDQFDLILNCASGIDGLNLSDYLSVLKVDKKFVSVGLPPIDDEFNVSPFTFLKQGASFGSSLLGSKAEVNIMLELAAKHNIRPWIEKVPISEENVAKALKRCFEGDVRYRFVFTEFDKAFGN SEQ ID ADH7atgactgttgacgcttcttctgttccagacaagttccaagggtttgcctccgacaagagagaaaactNO: 126 (Nucleic Acidgggaacacccaaagttgatctcctacgacagaaagcaactcaatgaccacgacgttgtcttgaa Seq.)gaacgagacctgtggtttgtgttactcggacatccacaccttgcgttccacgtggggaccatacggcaccaatgagcttgtcgttggccacgaaatctgtggtaccgtcattgctgtcggtccaaaggtcactgagttcaaggtcggtgacagagccggtattggtgctgcctcttcgtcttgtcgtcactgttccagatgtacccacgataacgagcaatactgtaaggaacaagtctccacttacaattctgttgatccaaaggccgctggttacgtcaccaagggtggttactcctcccactccatcgctgacgaattgtttgtcttcaaggttccagatgacttgccattcgagtacgcttccccattattctgtgctggtatcacaactttctccccattgtaccgtaacttggttgggtccgataaagacgccactggtaagaccgttggtatcattggtgttggtggtcttggtcaccttgccatccagtttgcgtctaaagctttgaacgctaaggtcgttgctttctccagatcctcctccaagaaggaagaagctctcgaattgggtgctgctgagtttgtcgccaccaacgaagacaagaactggaccagcagatacgaggaccaattcgacctcatcttgaactgtgcgagcggtatcgatggcttgaacttgtctgactacttgagtgtcttgaaagtcgacaagaagtttgtctctgttggtttgccaccaatcgacgacgagttcaacgtctctcctttcactttcttgaagcaaggtgccagtttcggtagttccttgttgggatccaaggctgaagtcaacatcatgttggaattggctgccaagcacaacatcagaccatggattgaaaaggtcccaatcagtgaggaaaacgtcgccaaggctttgaagagatgttttgaaggtgatgtcagatacagattcgtcttcactgagtttgacaaagcttttggcaat SEQ ID ADH8 (AminoMSVPTTQKAVIFETNGGKLEYKDVPVPVPKPNELLVNVKYSGVCHSD NO: 127 Acid Seq.)LHVWKGDWPIPAKLPLVGGHEGAGVVVGMGDNVKGWKVGDLAGIKWLNGSCMNCEFCQQGAEPNCSRADMSGYTHDGTFQQYATADAVQAAKIPEGADMASIAPILCAGVTVYKALKNADLLAGQWVAISGAGGGLGSLGVQYAKAMGYRVLAIDGGDERGEFVKSLGAEVYIDFLKEQDIVSAIRKATGGGPHGVINVSVSEKAINQSVEYVRTLGKVVLVSLPAGGKLTAPLFESVARSIQIRTTCandidaGNRKDTTEAIDFFVRGLIDCPIKVAGLSEVPEIFDLMEQGKILGRYVVDTSK SEQ ID ADH8AtgtccgttccaactactcagaaagctgttatctttgaaaccaatggtggcaagttagaatacaaagNO: 128 (Nucleic AcidacgtgccggtccctgtccctaaacccaacgaattgcttgtcaacgtcaagtactcgggtgtgtgtcatSeq.)tctgacttgcatgtctggaaaggcgactggcccattcctgccaagttgcccttggtgggaggtcacgaaggtgctggtgtcgttgtcggcatgggtgacaacgtcaagggctggaaggtgggggacttggctggtatcaagtggttgaatggttcgtgtatgaactgtgagttttgccaacagggcgcagaacctaactgttcaagagccgacatgtctgggtatacccacgatggaactttccaacaatacgccactgctgatgctgtccaagctgccaagatcccagaaggcgccgacatggctagtatcgccccgatcttgtgcgctggtgtgaccgtgtacaaggctttgaagaacgccgacttgttggctggccaatgggtggctatctctggtgctggtggtggtttgggctccttgggtgtgcagtacgctaaagccatgggttacagagtgttggctatcgacggtggtgacgagagaggagagtttgtcaagtccttgggcgccgaagtgtacattgacttccttaaggaacaggacatcgttagtgctatcagaaaggcaactggtggtggtccacacggtgttattaacgtctcagtgtccgaaaaggcaatcaaccagtcggtggagtacgtcagaactttggggaaagtggttttagttagcttgccggcaggtggtaaactcactgctcctcttttcgagtctgttgctagatcaatccagattagaactacgtgtgttggcaacagaaaggatactactgaagctattgatttctttgttagagggttgatcgattgcccaattaaagtcgctggtttaagtgaagtgccagagatttttgacttgatggagcagggaaagatcttgggtagatatgtcgttgatacgtcaaag SEQ ID SFA1 (AminoMSESTVGKPITCKAAVAWEAGKPLTIEDVTVAPPKAHEVRIKILYTGVC NO: 129 Acid Seq.)HTDAYTLSGVDPEGAFPVILGHEGAGIVESVGEGVTTVKPGDHVIALYTPECGECKFCKSGKTNLCGKIRATQGKGVMPDGTPRFTCKGKELIHFMGCSTFSQYTVVTDISVVAINDKAELDKACLLGCGITTGYGAATITANVQKGDNVAVFGGGAVGLSVLQGCKEREAAQIILVDVNNKKKEWGEKFGATAFINPLELPKGVTIVDKLIEMTDGGCDFTFDCTGNVNVMRDALEACHKGWGTSVIIGVAAAGKEISTRPFQLVTGRVWKGAAFGGVKGRSQLPGIVEDYMVGKLKVEEFITHRKPLEQINEAFEDMHAGDCIRAVVNMW SEQ ID SFA1 (NucleicatgtctgaatcaaccgttggaaaaccaatcacctgtaaagccgctgttgcctgggaagcaggcaaNO: 130 Acid Seq.) nogcctttgaccatcgaagacgtcactgttgctccaccaaaggcccacgaagtgcgtatcaagatcttintronsgtacactggtgtctgtcacactgatgcctacaccttgagtggtgttgatccagagggtgccttcccagtcatcttgggacacgaaggtgccggtattgttgaaagtgttggtgaaggtgtcaccactgtcaagccaggcgaccacgtcattgcattatacactccagaatgtggtgagtgtaagttctgtaaatcgggtaagaccaacttgtgtggtaaaatcagagctacccaaggcaaaggtgtgatgccagacggaactccaagattcacttgcaagggcaaagaattgattcactttatgggatgctccaccttctcccaatacaccgttgtcactgacatttccgtcgtggccatcaacgacaaagccgaacttgacaaggcttgtttgttgggatgtggtatcactactggttatggtgctgccaccatcactgccaatgttcaaaagggtgacaatgtcgcagttttcggtggtggtgctgtcggattgtccgtcctccaaggatgtaaagaaagagaagctgcccaaatcattttggttgatgtcaacaacaagaagaaggaatggggtgaaaagttcggtgccactgcttttatcaacccattagaattaccaaaaggcgtcactattgttgacaagttgattgaaatgactgacggtggttgtgactttacttttgactgtaccggtaatgtcaatgttatgagagatgccttggaagcttgtcataagggttggggtacttcagtcatcatcggtgttgccgctgccggtaaggaaatctccaccagaccattccaattggtcactggtagagtctggaagggtgctgctttcggtggcgtcaagggtagatcccaattgccaggaatcgttgaagactacatggttggtaagttgaaggttgaagagtttatcacccacagaaaaccattggaacaaatcaatgaagcatttgaagacatgcatgctggtgattgtattagagctgttgtcaacatgtgg SEQ ID FAO1,atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacggNO: 131 Candidagatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaa(Nucleic Acidgtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgt Seq.)ctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaagccaagttgtag SEQ ID FAO1,MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEY NO: 132 CandidaVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALT (Amino AcidNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLAN Seq.)ELHLKAIHYPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGlAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTKAKL SEQ ID FAO1Δpts1,atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacggNO: 133 Candidagatcatccacgaaaccaccgtcgaccaaatcaaagacgttattgctcctgacttccctgctgacaa(Nucleic Acidgtacgaagagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccgt Seq.)ctacaacacagtcaacgcaaacaccacggacgcaatccaccagttcattatcttgaccaatgttttggcatccagggtcttggctccagctttgaccaactcgttgacgcctatcaaggacatgagcttggaagaccgtgaaaaattgttggcctcgtggcgcgactccccaatcgctgccaaaaggaagttgttcaggttggtttctacgcttaccttggtcacgttcacgagattggccaatgagttgcatttgaaagccattcattatccaggaagagaagaccgtgaaaaggcttatgaaacccaggagattgacccttttaagtaccagtttttggaaaaaccgaagttttacggcgctgagttgtacttgccagatattgatgtgatcattattggatctggtgccggtgctggtgttgtggcccacactttggccaacgatggcttcaagagtttggttttggaaaagggcaaatactttagcaactccgagttgaactttgatgacaaggacggcgttcaagaattataccaaagtggaggtactttgactacagtcaaccaacagttgtttgttcttgctggttccacttttggtggcggtaccactgtcaattggtcagcctgtcttaagacgccattcaaggtgcgtaaggaatggtatgatgagtttggtgttgactttgctgctgatgaagcatacgataaagcgcaggattatgtttggcagcaaatgggagcttctaccgaaggcatcacccactctttggctaacgagattattattgaaggtggtaagaaattaggttacaaggccaaggtattagaccaaaacagcggtggtcatcctcagcacagatgcggtttctgttatttgggctgtaagcacggtatcaagcagggttctgttaataactggtttagagacgcagctgcccacggttcccagttcatgcaacaggttagagttttgcaaatacttaacaagaaggggatcgcttacggtatcttgtgtgaggatgttgtaaccggcgccaagttcaccattactggccccaaaaagtttgttgttgctgccggtgctttgaacactccatctgtgttggtcaactccggcttcaagaacaagaacatcggtaagaacttaactttgcacccagtttctgtcgtgtttggtgattttggcaaagacgttcaagcagaccacttccacaactccatcatgactgccctttgttcagaagccgctgatttagacggcaagggccatggatgcagaattgaaaccatcttgaacgctccattcatccaggcttcattcttaccatggagaggtagtaacgaggctagacgagacttgttgcgttacaacaacatggtggcgatgttgctccttagtcgtgacaccaccagtggttccgtttctgctcatccaaccaaacctgaagctttggttgtcgagtacgacgtgaacaagtttgacagaaactcgatcttgcaggcattgttggtcactgctgacttgttgtatatccaaggtgccaagagaatccttagtccacaggcatgggtgccaatttttgaatccgacaagccaaaggataagagatcaatcaaggacgaggactatgtcgaatggagagccaaggttgccaagattcctttcgacacctacggctcaccttatggttcggcacatcaaatgtcttcttgccgtatgtcaggtaagggtcctaaatacggtgctgttgacaccgatggtagattgtttgaatgttcgaatgtttatgttgccgatgcaagtcttttgccaactgcaagcggtgccaaccctatggtcaccaccatgactcttgccagacatgttgcgttaggtttggcagactccttgaagaccaaatag SEQ ID FAO1Δpts1,MAPFLPDQVDYKHVDTLMLLCDGIIHETTVDQIKDVIAPDFPADKYEEY NO: 134 CandidaVRTFTKPSETPGFRETVYNTVNANTTDAIHQFIILTNVLASRVLAPALT (Amino AcidNSLTPIKDMSLEDREKLLASWRDSPIAAKRKLFRLVSTLTLVTFTRLAN Seq.)ELHLKAIHYPGREDREKAYETQEIDPFKYQFLEKPKFYGAELYLPDIDVIIIGSGAGAGVVAHTLANDGFKSLVLEKGKYFSNSELNFDDKDGVQELYQSGGTLTTVNQQLFVLAGSTFGGGTTVNWSACLKTPFKVRKEWYDEFGVDFAADEAYDKAQDYVWQQMGASTEGITHSLANEIIIEGGKKLGYKAKVLDQNSGGHPQHRCGFCYLGCKHGIKQGSVNNWFRDAAAHGSQFMQQVRVLQILNKKGlAYGILCEDVVTGAKFTITGPKKFVVAAGALNTPSVLVNSGFKNKNIGKNLTLHPVSVVFGDFGKDVQADHFHNSIMTALCSEAADLDGKGHGCRIETILNAPFIQASFLPWRGSNEARRDLLRYNNMVAMLLLSRDTTSGSVSAHPTKPEALVVEYDVNKFDRNSILQALLVTADLLYIQGAKRILSPQAWVPIFESDKPKDKRSIKDEDYVEWRAKVAKIPFDTYGSPYGSAHQMSSCRMSGKGPKYGAVDTDGRLFECSNVYVADASLLPTASGANPMVTTMTLARHVALGLADSLKTK SEQ ID ALD1,MLSRVLFKTKPRVPTKSITAMAIRNKSIVTLSSTTSTYPTDHTTPSTEP NO: 135 CandidaYITPSFVNNEFIKSDSNTWFDVHDPATNYVVSKVPQSTPEELEEAIAS (Amino AcidAHAAFPKWRDTSIIKRQGIAFKFVQLLRENMDRIASVIVLEQGKTFVDA Seq.)QGDVTRGLQVAEAACNITNDLKGESLEVSTDMETKMIREPLGVVGSICPFNFPAMVPLWSLPLVLVTGNTAVIKPSERVPGASMIICELAAKAGVPPGVLNIVHGKHDTVNKLIEDPRIKALTFVGGDKAGKYIYEKGSSLGKRVQANLGAKNHLVVLPDAHKQSFVNAVNGAAFGAAGQRCMAISVLVTVGKTKEWVQDVIKDAKLLNTGSGFDPKSDLGPVINPESLTRAEEIIADSVANGAVLELDGRGYRPEDARFAKGNFLGPTILTNVKPGLRAYDEEIFAPVLSVVNVDTIDEAIELINNNKYGNGVSLFTSSGGSAQYFTKRIDVGQVGINVPIPVPLPMFSFTGSRGSFLGDLNFYGKAGITFLTKPKTITSAW KTNLIDDEILKPSTSMPVQQSEQ ID ALD1,atgttatccagagttcttttcaagactaaaccaagagttcctactaaatcaatcaccgccatggccatNO: 136 Candidacagaaacaaatccatcgtgactttatcctccaccacctccacatacccaaccgaccacacgaccc(Nucleic Acidcgtccacggagccatacatcacgccatccttcgtgaacaacgagttcatcaagtcggactccaac Seq.)acctggttcgacgtgcacgacccggccacgaactacgtcgtgtccaaggtgccacagtcgacgccggaggagttggaagaagcaatcgcgtcggcccacgccgcgttccccaaatggcgcgacaccagcatcatcaagcgtcaagggatcgcgttcaagtttgtgcagttgttgcgcgagaacatggacagaatcgcaagcgtcattgtcttggaacaaggtaagacgtttgtcgatgcccagggtgacgtgactagaggattacaggttgctgaggctgcgtgcaacatcactaacgacttgaaaggtgagtcgttggaagtgtctactgatatggagaccaagatgattagagaacctttgggtgttgtgggatccatctgtccttttaacttcccagctatggtcccattgtggtctttgcctttggttttggtcacgggtaacaccgctgtgattaagccttccgagagagtcccgggcgcaagtatgattatttgtgaattggccgccaaggcaggtgttccacctggtgtgttgaacattgtccacggtaagcacgacaccgtcaacaagttgatcgaggacccaagaatcaaggcattgacttttgtcggtggtgacaaggccggtaagtacatttacgaaaagggttccagtttgggcaagagagtgcaggccaacttgggtgctaagaaccacttggttgtgttgccagacgcacacaagcagagttttgtcaatgccgtcaacggtgccgctttcggtgctgctggacagagatgtatggctatttctgtcttggtcaccgtgggtaagaccaaggaatgggtgcaggatgtcatcaaggacgccaagttgttgaacaccggaagtggatttgacccaaagagtgacttgggtccagtcatcaacccagagtccttgactcgtgctgaagaaatcattgctgattccgtggccaacggtgccgtgttggaattggacggaagaggatacagaccagaagacgccagattcgccaagggtaacttcttgggtccaaccatcttgaccaacgtcaagccgggcttgagagcatacgacgaggagattttcgctcctgttttgtcggtggttaacgtcgacaccattgacgaagccattgagttgatcaacaataacaagtacggtaacggtgtttcattatttacttcctccggtggctcagcccagtatttcactaagagaatcgatgtcggccaagtcggtatcaatgtcccaatccctgttccattgcctatgttctccttcactggttccagaggctccttcttgggtgacttgaacttctacggtaaggccggtatcaccttcttgaccaagccaaagaccatcactagtgcctggaagaccaacttgattgatgacgagatcttgaaaccatctacctcgatgcctgtccaacag SEQ ID ALD5,MSLPVVTKLTTPKGLSYNQPLGLFINNEFVVPKSKQTFEVFSPSTEEKI NO: 137 CandidaTDVYEALAEDVDVAAEAAYAAYHNDWALGAPEQRAKILLKLADLVEE (Amino AcidHAETLAQIETWDNGKSLQNARGDIGFTAAYFRSCGGWADKNTGDNI Seq.)NTGGTHLTYTQRVPLVCGQIIPWNASTLMASWKLGPVIATGGTTVLKSAEATPLAVLYLAQLLVEAGLPKGVVNIVSGFGTTAGSAIASHPKIDKVAFTGSTNTGKIIMKLAAESNLKKVTLELGGKSPHIVFNDADLDRAVSYLVAAIFSNSGETCAAGSRVLVQSGVYDEVVAKFKKGAEAVKVGDPFDEETFMGSQVNEVQLSRILQYIESGKEQGATVVTGGGRAGDKGYFVKPTIFADVHKDMTIVKEEIFGPVVSVVKFDTIEEAIALANDSEYGLAAGIHTTNISTGVTVANRIKSGTVWVNTYNDLHPMVPFGGFGASGIGREMGAEV MKEYTEVKAVRIKLT SEQ IDALD5,atgtctttgccagtcgtcaccaaactcactactcctaagggtctctcctacaaccaaccattaggtttgNO: 138 Candidattcatcaacaacgagttcgttgttccaaaatccaagcaaaccttcgaagtcttctccccttccaccga(Nucleic Acidagagaagatcaccgatgtctacgaagctttagccgaagatgtcgacgttgctgccgaagcagctt Seq.)acgccgcctaccacaacgactgggcccttggtgctccagaacaaagagccaagatcttgctcaagttggccgacttggtcgaagaacacgccgagaccttggcccagatcgaaacctgggacaacggtaagtccttgcagaacgccagaggcgatatcggattcactgccgcttactttagatcctgtggtggatgggccgacaagaacaccggtgacaacatcaacaccggtggcacccaccttacttacacccagagagtcccattggtgtgtggtcaaatcatcccttggaacgcaagtaccttgatggccagttggaagcttggtcccgttatcgctaccggtggtaccactgtgcttaaatcagctgaagctaccccattagctgtcttgtacctcgcccaattgttagttgaagccggtcttccaaagggtgtcgttaacattgtttccggtttcggtaccactgccggttccgctatcgctagccatccaaagatcgacaaggtcgcctttactggttccaccaacaccggtaagatcatcatgaagttggctgcggagtccaacttgaagaaggtcactttggaattgggtggtaagtccccacacattgttttcaacgacgctgacttggaccgcgccgtcagctacttggttgctgccattttcagtaactccggcgagacctgtgctgccggatcccgtgtcttggtgcaatccggtgtctacgacgaagttgttgctaagttcaagaagggcgccgaggccgttaaagttggtgacccattcgacgaagaaaccttcatgggttcccaagtcaacgaagtccaattgtctagaatcttgcaatacatcgagctgggtaaggaacaaggtgccactgttgtcaccggtggtggtagagccggggacaagggttacttcgtcaagccaactattttcgccgacgttcacaaggacatgactatcgtcaaggaagaaatctttggtcctgttgtctccgtcgtcaaattcgataccattgaagaagctatcgctttggctaacgactccgaatacggtttggccgctggtatccacaccactaacatcagcaccggtgtcaccgtcgctaacagaatcaagtccggtactgtctgggtcaacacttacaatgacttgcaccccatggttccattcggtggtttcggcgcttctggtatcggcagagaaatgggtgcagaagtcatgaaggaatacaccgaagttaaggctgttagaattaagctcact SEQ ID HFD1,MSKSYKLPKSSKISPIVKGKTSAKSKSSSKTPSPPSGSPPTSARIAAPE NO: 139 CandidaLEPVEQTSDNELPATKVAVRRSSSASSKSTNGSAAATSAAAANAAAP (Amino AcidQKTPVEAKPAPKPEPVQSKGNDNDSDDSKLDTAESYVDVKKETEALV Seq.)ESKSVASTVDDTSVLQYTPLSEIPGGVKRVVDGFHTGKTHPLEFRLKQLRNLYFAVRDNQEAICDALSKDFHRVSSETRNYELVTGLNELLYTMSQLHKWSKPLPVDELPLNLMINPTYVERIPVGTVLVIAAFNYPLFVSISPIAGAIAAGNTVVFKPSELTPHFSKLFTDLMAKALDPDVFYAVNGSVPETTELLNQKFDKIIYTGSETVGKIIAKKAAETLTPVILELGGKSPAFVLDDVADKDLPIVARRIAWGRYANAGQTCIGVDYVLVAKSKHDKFIKALRDVIEKEFFPNVDANSNFTHLIHDRAFHKMKNIIDKTTGKIIIGGQMDSASRYVSPTVIDDATWDDSSMQEEIFGPILPVLTYTDLTDACRDVVSHHDTPLAEYIFTSGSTSRKYNSQINTIATIIRSGGLVINDVLMHIALHNAPFGGIGKSGHGAYHGEFSYRAFTHERTVLEQNLWNDWIIKSRYPPYSNKKDRLVASSQGKYGGRVWFGRQGDVKIDGPSTFFSAWTNVLGVAGVVCDFIG ASL SEQ ID HFD1,atgagtaagtcatacaagttgccaaaatcatctaagatctcgcctatcgtaaagggtaagacctcgNO: 140 Candidagcaaaatcaaaaagtagctcaaaaacaccatcaccaccactgggatccccaccaacatcagc(Nucleic Acidcaggatcgctgcgcctgaattggaaccggtcgaacaaacatccgacaacgagctcccagctact Seq.)aaagtggccgttcgcagaagcagcagcgcttcatcgaagtcaaccaacgggtccgcggctgccacttctgccgctgctgccaatgctgctgccccacaaaaaactccagttgaagcaaagccagcccctaagccagagccagttcagtccaagggtaacgacaacgactccgatgactccaagttagacaccgctgaatcatatgtcgacgtgaagaaagaaaccgaagctctcgttgagtcaaagtcggttgcttcaacagtcgatgatacttctgtcttgcagtacaccccgttatctgagatccctggcggcgtcaagagagttgtcgatgggttccacaccggcaagacccacccattggagtttagattgaagcaattgagaaacttgtacttcgccgtgagggataaccaagaagccatctgtgacgccttgagcaaggatttccaccgtgtctcatccgaaactagaaactatgaattggttaccgggttgaatgaattgttgtacacgatgtcgcaattgcacaagtggagcaagccattgccagtggatgagttgccattgaacttgatgatcaaccctacttatgttgaaagaatcccagttggtacggttttggtcatcgccgctttcaactatccattgtttgtttcgatttcccctattgccggtgccattgctgctggtaacactgttgtgttcaagccatctgaattgactccgcacttttccaagttgttcactgatttgatggctaaagcgttggacccagatgtcttttatgccgtgaacggttccgtgcctgaaactaccgagttgttgaaccagaagttcgacaagatcatttacaccggtagtgaaactgttggtaagatcattgccaagaaggcggccgagacgttgacgccagtgattttggaacttggaggcaaatcgccagcctttgttttggacgacgttgccgacaaagacttgccaattgttgcgcgtcgtattgcttggggaagatatgctaatgctggacagacctgtattggtgttgattatgtgttggttgcaaagtccaaacacgacaagttcatcaaggctttgagggatgttattgagaaagagttcttccccaatgttgacgctaacagcaactttacccatttgatccacgacagggcattccacaaaatgaagaacatcattgacaaaaccacagggaagataatcatcggtggtcaaatggatagtgcttccaggtatgtctcaccaactgtgattgacgatgctacctgggacgactcgtccatgcaggaggaaatatttggccctatcttgccggttctcacttatactgaccttactgatgcatgtcgtgacgttgtttctcaccatgatactccgttggctgaatacatcttcaccagcgggtccacttcaagaaagtacaactcgcagatcaacaccattgctactatcatcagatccggtgggttagtcataaatgatgtgttgatgcacattgccttgcacaacgctccgttcggtggtattggtaaatctggacatggtgcctaccacggagaattctcctacagggcttttacccacgagagaaccgttcttgaacaaaacttgtggaatgactggatcattaagtcaagatacccaccatattccaataagaaagacaggttggttgctagttcacaaggcaagtatggtggaagagtttggttcggacgtcaaggtgatgtcaagattgacggaccatccacgttcttctcggcatggaccaatgtccttggtgtagctggtgtagtgtgtgattttattggtgcctctttg SEQ ID HFD2,MSPPSKLEDSSSATTAADTLGDSWYTKVSDIAPGVQRLTESFHRDQK NO: 141 CandidaTHDIQFRLNQLRNLYFAVQDNADALCAALDKDFYRPPSETKNLELVG (Amino AcidGLNELVHTISSLHEWMKPEKVTDLPLTLRSNPIYIERIPLGVVLIISPFNY Seq.)PFFLSFSAVVGAIAGGNAVVLKGSELTPNFSSLFSKILTKALDPDIFFAVDGAIPETTELLEQKFDKIMYTGNNTVGKIIAKKAAETLTPVILELGGKSPAFILDDVKDKNLEVIARRIAWGRFTNAGQTCandidaAVDYVLVPTKLHKKFIAALTKVLSQEFYPNLTKDTKGYTHVIHDRAFNNLSKIISTTKGDIVFGGDTDAATRFIAPTVIDNATWEDSSMKGEIFGPILPVLTYDKLTTAIRQVVSTHDTPLAQYIFTSGSTSRKYNRQLDQILTGVRSGGVIVNDVLMHVALINAPFGGVGDSGYGSYHGKFSFRSFTHERTTMEQKLWNDGMVKVRYPPYNSNKDKLIQVSQQNYNGKVWFDRNGDVPVNGPGALFSAWT TFTGVFHLLGEFITNKQ SEQ IDHFD2, atgtccccaccatctaaattagaagactcctcctccgcaaccaccgctgccgatacccttggcgacNO: 142 Candidatcctggtacaccaaagtgtccgacattgcgcctggcgtgcagagattgaccgagtcattccacagg(Nucleic AcidgatcaaaagacgcacgacattcagttccgcttgaaccaattgcgtaacctttactttgcggtccaggSeq.) acaatgccgacgcgctctgtgctgccttggacaaggacttctaccgtccccccagtgaaaccaagaacttggaactcgtgggtggcttgaatgagttggtgcacaccatttcgagcttgcatgagtggatgaagccggaaaaagtcacggatttgccacttactttgaggtcaaacccgatttatattgaaagaatcccattgggggtcgtgttgatcatctcgcctttcaactaccctttcttcttgtcgttttcggccgtcgtgggtgcgattgctggtggtaacgcggttgttttgaagggctctgagttgacgccaaacttctccagtttgttctcaaagatcttgactaaggctttggaccctgatattttctttgcagtcgatggtgctatccctgagacgaccgagttgttggaacaaaagtttgacaagatcatgtatactggtaacaacaccgtgggtaagattattgccaagaaggctgctgagaccttgacgccagttatcttggaattgggtggtaagtcgccagctttcatcttggacgacgtcaaggataaaaacttggaagtcatcgccagaagaatcgcatggggtagattcaccaacgccggtcaaacctgtgttgctgtcgactacgtcttggttccaaccaaactccacaagaagttcattgctgcgttgaccaaggtcttgagtcaagaattctaccctaacttgaccaaagacaccaagggctacacccacgtcatccacgaccgtgcattcaacaatttgtccaagatcatcagcaccaccaagggtgacattgtctttggcggcgacaccgatgccgccacccgcttcatcgcccccaccgtcatcgacaacgccacctgggaggattcttccatgaagggcgaaatctttggtcccatcttgcccgtcttgacctacgacaagctcaccaccgccatcaggcaagttgtgtccacgcacgacacgccattagcgcagtacatcttcaccagcgggtccacatcccgcaagtacaaccgccagctcgaccagatcttgactggtgtccggtccgggggtgtgattgtcaacgatgtcttgatgcacgttgcgttgatcaatgcgccatttggcggcgttggtgactccgggtacggctcgtaccacggcaagttctcgttccgcagcttcacgcacgaacgtaccaccatggagcagaagttgtggaacgacgggatggtcaaggtcagataccctccttataactccaacaaggacaagttgatccaggtctcccagcagaactacaacggcaaggtctggttcgatagaaacggcgacgtgcctgtgaatggaccaggtgcgttgtttagcgcttggactacgttcactggtgtcttccatttgcttggtgagttcatcactaataagcaataga SEQ ID MAE1 (non-MVSSTATASATSGEMRTVKTPVGIKAAIESLKPKATRVSMDGPVECPL NO: 143 mitochondrial),TDFALLNSPQFNKGSAFSLEERKSFKLTGLLPSQVNTLDEQVERAYR CandidaQFTYLKTPLAKNDFCTSMRLQNKVLYYELVRRNIREMLPIIYTPTEGDA (Amino AcidIASYSDRFRKPEGCFLDINDPDNIDERLAAYGENKDIDYIVMSDGEGIX Seq.)XXSDRFRKPEGCFLDINDPDNIDERLAAYGENKDIDYIVMSDGEGILGIGDQGVGGIRIAIAKLGLMTLCGGIHPARVLPITLDVGTNNDRLLNDDLYMGNKFPRVRGERYWDFVDKVIHAITKRFPSAVMHYEDFGVTTGRDMLHKYRTALPSFNDDIQGTGAVVMASITAALKFSNRSLKDIEVLIYGAGSAGLGIADQITNHLVSHGATPEQARSRIHCMDRYGLITTESNNASPAQMNYADKASDWEGVDTSSLLACandidaEKVKPTVLVGCSTQAGAFTEEVVKTMYKYNPQPIIFPLSNPTRLHEAVPADLMKWTDNNALIATGSPFEPVDGYYISENNNCFTFPGIGLGAVLSRCSTISDTMISAAVDRLASMSPKMENPKNGLLPRLEEIDEVSAHVATAVILQSLKEGTARVESEKKPDGGYVEVPRDYDDCLKWVQSQMWKPVYRPYIKVEYVSNIHTYQY SEQ ID MAE1 (non-atggtctcctccacggccaccgcatccgccacgtcaggggaaatgcgtaccgtcaagaccccag NO: 144mitochondrial),tggggatcaaggcggccatcgaatcattaaaaccaaaagctactagagtctccatggacggacc Candidatgtcgaatgcccattgaccgatttcgccttgttgaactcccctcaattcaacaaaggttcggcattttctt(Nucleic Acidtggaagaaaggaaaagtttcaagttgaccgggctcctcccttctcaagtcaacactttggatgaac Seq.)aggttgaaagagcctatagacaattcacatacttgaagaccccattggccaagaacgatttctgcacgtctatgagattgcagaacaaagtgctttactacgagttggttagaagaaatatccgtgagatgttgcccatcatctacaccccaaccgaaggggacgccatcgccagttattccgacaggttcagaaaaccagagggctgtttcttggatatcaacgaccccgacaacatcgatgagagattagctgcctatggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcnnnnnnnnctccgacaggttcagaaaaccagagggctgcttcttggacatcaatgacccagacaacatcgacgagagattggctgcctatggggagaacaaagacatagattacattgtcatgagtgacggagaaggtatcctcggtattggagaccaaggcgtcggtggtatcagaattgccattgctaaattggggttgatgaccctttgtggtggtattcacccggccagagttttgcccatcactttggatgttggtacaaataacgacaggttgttgaatgatgatttgtacatgggcaacaagttccctagagtcagaggagaaagatactgggactttgtcgataaggtcatacacgcaattacgaaacggttcccaagtgccgtgatgcattacgaagatttcggagtcacaactggtagggacatgttgcacaagtaccgtacggctcttccttctttcaacgacgacatccaaggtaccggtgcagttgtcatggcatcgatcacagctgccttgaagttctccaaccgtagcctaaaggacatcgaggttttgatttacggtgccggctcagctggtttaggtattgctgaccagatcaccaaccacttggtcagccacggcgctactccagaacaagccagatctaggatccattgtatggaccgttatgggttgatcacaactgaatccaacaacgccagtcctgctcaaatgaactacgccgacaaggcatctgattgggaaggtgtcgatacctcgagtctacttgcctgtgttgagaaagtcaaaccaactgtcttggttgggtgttccactcaggcaggtgcattcaccgaagaggttgtcaaaaccatgtacaagtacaacccacagccaattattttcccattgtccaaccctaccagattgcatgaagccgtgccggctgatttgatgaaatggaccgacaacaacgcgttgattgccaccggttctccatttgaacctgtcgatggctactacatttccgaaaacaacaactgtttcaccttcccaggtattgggttgggtgctgtcttgtccagatgtagcaccatttcggataccatgatttctgccgccgttgatagattggcttcgatgtcgccaaagatggagaacccaaagaacggattgttgcctagattggaagaaatcgacgaagtcagtgcccatgttgccacggctgttatcttgcaatctttgaaggaaggcaccgctagagtcgaaagcgagaagaagccagacggtggttacgttgaagttccaagagactatgatgattgtcttaagtgggtgcaatcacaaatgtggaagccagtgtacagaccatacatcaaggttgagtacgtttcgaatattcacacctatcaatat SEQ IDPYC2, Sc MPESRLQRLANLKIGTPQQLRRTSIIGTIGPKTNSCEAITALRKAGLNII NO: 145(Amino Acid RLNFSHGSYEFHQSVIENAVKSEQQFPGRPLAIALDTKGPEIRTGRTL Seq.)NDQDLYIPVDHQMIFTTDASFANTSNDKIMYIDYANLTKVIVPGRFIYVDDGILSFKVLQIIDESNLRVQAVNSGYIASHKGVNLPNTDVDLPPLSAKDMKDLQFGVRNGIHIVFASFIRTSEDVLSIRKALGSEGQDIKIISKIENQQGLDNFDEILEVTDGVMIARGDLGIEILAPEVLAIQKKLIAKCNLAGKPVICATQMLDSMTHNPRPTRAEVSDVGNAVLDGADCandidaMLSGETAKGDYPVNAVNIMAATALIAESTIAHLALYDDLRDATPKPTSTTETVAAAATAAILEQDGKAIVVLSTTGNTARLLSKYRPSCPIILVTRHARTARIAHLYRGVFPFLYEPKRLDDWGEDVHRRLKFGVEMARSFGMVDNGDTVVSI QGFKGGVGHSNTLRISTVGQEF*SEQ ID PYC2, Sc ATGCCAGAGTCCAGATTGCAGAGACTAGCTAATTTGAAAATAGGAA NO: 146(Nucleic Acid CTCCGCAGCAGCTCAGACGCACCTCCATAATAGGTACCATTGGGC Seq.)CCAAGACAAATAGCTGCGAGGCCATTACTGCTCTGAGAAAAGCTGGTTTGAACATCATTCGATTGAACTTTTCCCATGGCTCCTACGAATTCCATCAATCAGTAATCGAAAATGCTGTGAAATCGGAACAGCAATTCCCTGGCAGGCCGCTCGCCATTGCCCTGGATACCAAGGGTCCCGAGATCAGAACAGGTCGCACGTTAAATGACCAAGATCTTTATATCCCCGTAGACCACCAAATGATCTTTACCACTGACGCAAGTTTTGCAAACACCTCCAATGATAAAATCATGTATATAGACTATGCTAACCTGACAAAAGTTATCGTTCCGGGGAGATTTATATACGTGGACGACGGGATTCTCTCTTTTAAAGTGCTCCAAATCATTGACGAATCTAATTTAAGGGTGCAAGCGGTAAACTCGGGTTATATCGCATCTCATAAAGGTGTTAATCTGCCTAATACCGACGTTGATTTGCCCCCCTTGTCCGCCAAAGACATGAAGGACTTGCAATTCGGAGTCCGCAATGGCATTCACATCGTATTTGCCTCTTTCATAAGAACTTCAGAAGATGTGTTGTCTATCAGAAAAGCGTTGGGTTCTGAAGGGCAAGATATCAAGATTATATCCAAGATAGAAAACCAGCAAGGGTTGGATAATTTTGACGAAATCCTGGAAGTCACGGATGGTGTTATGATAGCGAGAGGCGATTTAGGAATTGAAATCCTGGCACCTGAAGTATTAGCCATTCAAAAAAAGCTGATTGCAAAATGTAATTTGGCGGGCAAACCTGTCATTTGCGCGACTCAGATGCTGGATTCAATGACACACAATCCGAGACCGACAAGGGCTGAAGTATCGGATGTGGGTAACGCTGTGTTGGATGGTGCTGATTGTGTTATGCTTTCTGGAGAAACGGCGAAGGGTGATTATCCGGTGAATGCAGTTAATATTATGGCGGCGACCGCTCTGATTGCTGAAAGTACTATCGCTCATTTGGCTCTTTATGACGATCTCAGAGACGCCACTCCCAAACCTACTTCCACTACGGAAACTGTAGCAGCTGCAGCTACCGCAGCAATCTTGGAGCAAGATGGTAAGGCCATCGTTGTATTATCTACTACAGGGAACACGGCAAGGCTACTGTCGAAGTATAGACCAAGCTGCCCTATCATATTAGTAACAAGACACGCAAGAACGGCAAGAATTGCGCATTTGTATAGAGGTGTTTTCCCATTTCTGTATGAACCGAAACGCCTAGACGACTGGGGTGAGGATGTTCATAGGCGCCTAAAGTTTGGTGTTGAAATGGCGAGGTCTTTCGGAATGGTGGACAACGGTGATACTGTTGTTTCCATTCAAGGATTCAAAGGAGGAGTCGGCCATTCCAATACCTTACGCATT TCTACTGTTGGTCAAGAATTCTAGSEQ ID FAT1 S244A,atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattNO: 147 ATCC20336gccgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccag(Nucleic Acidcagaggtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaag seq.)cgttggcgttcccaagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgacgatcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttgaagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattgtcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagccattgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcagagataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttgtttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaaccgatactgactcctccgcttgtgcattgatttacaccgcgggtaccaccggtttgccaaaagccggtatcatgtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagcagaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctgtaccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcagaaatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaaaggtgacgcgtggtacagatccggtgacttgttgaagatggacgaggacaaattgttgtactttgtcgacagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattgatgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggtaaagccaaattgtag SEQ ID FAT1 S244A,msgleiaaaailgsqlleakyliaddvslaktvavnalpylwkasrgkasywyffeqsvfknpnnkaNO: 148 ATCC20336lafprprknaptpktdaegfqiyddqfdleeytykelydmvlkysyilkneygvtandtigvscmnk(Amino Acidplfivlwlalwnigalpaflnfntkdkplihclkivnasqvfvdpdcdspirdteaqireelphvqinyidseq.)efalfdrlrlkstpkhraedktrrptdtdssacaliytagttglpkagimswrkafmasvffghimkidsksnvltamplyhstaamlglcptlivggCandidasvsqkfsatsfwtqarlcgathvqyvgevcryllnskphpdqdrhnvriaygnglrpdiwsefkrrfhiegigefyaatespiattnlqygeygvgacrkygslislllstqqklakmdpedeseiykdpktgfcteaaynepgellmrilnpndvqksfqgyygnksatnskiltnvfkkgdawyrsgdllkmdedkllyfvdrlgdtfrwksenvsatevenelmgskalkqsvvvgvkvpnhegracfavceakdelsheeilklihshvtkslpvyaqpafikigtieashnhkvpknqfknqklpkgedgkdliywlngdkyqelteddwslictgkakl SEQ ID FAT1 D495A,atgtcaggattagaaatagccgctgctgccatccttggtagtcagttattggaagccaaatatttaattNO: 149 ATCC20336gccgacgacgtgctgttagccaagacagtcgctgtcaatgccctcccatacttgtggaaagccag(Nucleic Acidcagaggtaaggcatcatactggtactttttcgagcagtccgtgttcaagaacccaaacaacaaag seq.)cgttggcgttcccaagaccaagaaagaatgcccccacccccaagaccgacgccgagggattccagatctacgacgatcagtttgacctagaagaatacacctacaaggaattgtacgacatggttttgaagtactcatacatcttgaagaacgagtacggcgtcactgccaacgacaccatcggtgtttcttgtatgaacaagccgcttttcattgtcttgtggttggcattgtggaacattggtgccttgcctgcgttcttgaacttcaacaccaaggacaagccattgatccactgtcttaagattgtcaacgcttcgcaagttttcgttgacccggactgtgattccccaatcagagataccgaggctcagatcagagaggaattgccacatgtgcaaataaactacattgacgagtttgccttgtttgacagattgagactcaagtcgactccaaaacacagagccgaggacaagaccagaagaccaaccgatactgactcctccgcttgtgcattgatttacacctcgggtaccaccggtttgccaaaagccggtatcatgtcctggagaaaagccttcatggcctcggttttctttggccacatcatgaagattgactcgaaatcgaacgtcttgaccgccatgcccttgtaccactccaccgcggccatgttggggttgtgtcctactttgattgtcggtggctgtgtctccgtgtcccagaaattctccgctacttcgttctggacccaggccagattatgtggtgccacccacgtgcaatacgtcggtgaggtctgtcgttacttgttgaactccaagcctcatccagaccaagacagacacaatgtcagaattgcctacggtaacgggttgcgtccagatatatggtctgagttcaagcgcagattccacattgaaggtatcggtgagttctacgccgccaccgagtcccctatcgccaccaccaacttgcagtacggtgagtacggtgtcggcgcctgtcgtaagtacgggtccctcatcagcttgttattgtctacccagcagaaattggccaagatggacccagaagacgagagtgaaatctacaaggaccccaagaccgggttctgtaccgaggccgcttacaacgagccaggtgagttgttgatgagaatcttgaaccctaacgacgtgcagaaatccttccagggttattatggtaacaagtccgccaccaacagcaaaatcctcaccaatgttttcaaaaaaggtgacgcgtggtacagatccggtgccttgttgaagatggacgaggacaaattgttgtactttgtcgacagattaggtgacactttccgttggaagtccgaaaacgtctccgccaccgaggtcgagaacgaattgatgggctccaaggccttgaagcagtccgtcgttgtcggtgtcaaggtgccaaaccacgaaggtagagcctgttttgccgtctgtgaagccaaggacgagttgagccatgaagaaatcttgaaattgattcactctcacgtgaccaagtctttgcctgtgtatgctcaacctgcgttcatcaagattggcaccattgaggcttcgcacaaccacaaggttcctaagaaccaattcaagaaccaaaagttgccaaagggtgaagacggcaaggatttgatctactggttgaatggcgacaagtaccaggagttgactgaagacgattggtctttgatttgtaccggtaaagccaaattgtag SEQ ID FAT1 D495A,MsgleiaaaailgsqlleakyliaddvslaktvavnalpylwkasrgkasywyffeqsvfknpnnkaNO: 150 ATCC20336lafprprknaptpktdaegfqiyddqfdleeytykelydmylkysyilkneygvtandtigvscmnk(Amino Acidplfivlwlalwnigalpaflnfntkdkplihclkivnasqvfvdpdcdspirdteaqireelphvqinyidseq.)efalfdrltlkstpkhraedktrrptdtdssacaliytsgttglpkagimswrkafmasvffghimkidsksnvltamplyhstaamlglcptlivggcvsysqkfsatsfwtqarlcgathvqyvgevcryllnskphpdqdrhnvriaygnglrpdiwsefkrrfhiegigefyaatespiattnlqygeygvgacrkygslislllstqqklakmdpedeseiykdpktgfcteaaynepgellmrilnpndvqksfqgyygnksatnskiltnvfkkgdawyrsgallkmdedkllyfvdrlgdffrwksenvsatevenelmgskalkqsvvvgvkvpnhegracfavceakdelsheeilklihshvtkslpvyaqpafikigtieashnhkvpknqfknqklpkgedgkdliywlngdkyqelteddwslictgkakl SEQ ID SFA1 (NucleicatgtctgaatcaaccgttggaaaagtatgtcaccttaacaattgagtctcgtaattgctcgccattgggNO: 151 Acid Seq.)aaatcatcggctgtatttataatccagatctttcctaccctagtagttaccacagaacaattgcaacagenomicgaataaatactaacctttttgtcttagccaatcacctgtaaagccgctgttgcctgggaagcaggcaagcctttgaccatcgaagacgtcactgttgctccaccaaaggcccacgaagtgcgtatcaagatcttgtacactggtgtctgtcacactgatgcctacaccttgagtggtgttgatccagagggtgccttcccagtcatcttgggacacgaaggtgccggtattgttgaaagtgttggtgaaggtgtcaccactgtcaagccaggcgaccacgtcattgcattatacactccagaatgtggtgagtgtaagttctgtaaatcgggtaagaccaacttgtgtggtaaaatcagagctacccaaggcaaaggtgtgatgccagacggaactccaagattcacttgcaagggcaaagaattgattcactttatgggatgctccaccttctcccaatacaccgttgtcactgacatttccgtcgtggccatcaacgacaaagccgaacttgacaaggcttgtttgttgggatgtggtatcactactggttatggtgctgccaccatcactgccaatgttcaaaagggtgacaatgtcgcagttttcggtggtggtgctgtcggattgtccgtcctccaaggatgtaaagaaagagaagctgcccaaatcattttggttgatgtcaacaacaagaagaaggaatggggtgaaaagttcggtgccactgcttttatcaacccattagaattaccaaaaggcgtcactattgttgacaagttgattgaaatgactgacggtggttgtgactttacttttgactgtaccggtaatgtcaatgttatgagagatgccttggaagcttgtcataagggttggggtacttcagtcatcatcggtgttgccgctgccggtaaggaaatctccaccagaccattccaattggtcactggtagagtctggaagggtgctgctttcggtggcgtcaagggtagatcccaattgccaggaatcgttgaagactacatggttggtaagttgaaggttgaagagtttatcacccacagaaaaccattggaacaaatcaatgaagcatttgaagacatgcatgctggtgattgtattagagctgttgtcaacatgtgg SEQ ID ADH1-1short,MSANIPKTQKAVVFEKNGGELEYKDIPVPTPKANELLINVKYSGVCHT NO: 152 CandidaDLHAWKGDWPLATKLPLVGGHEGAGVVVGMGENVKGWKIGDFAGI (Amino AcidKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGSFEQYATADAVQ Seq.)AARIPAGTDLAEVAPILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGAFVKSLGAEAYIDFLKEKDIVSAVKKATDGGPHGAINVSVSEKAIDQSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGLSELPQ VFKLMEEGKILGRYVLDTSKSEQ ID ADH1-1short,atgtctgctaatatcccaaaaactcaaaaagctgtcgtctttgagaagaacggtggtgaattagaatNO: 153 Candidaacaaagatatcccagtgccaaccccaaaggccaacgaattgctcatcaacgtcaaatactcggg(Nucleic AcidtgtctgccacactgatttgcacgcctggaagggtgactggccattggccaccaagttgccattggttSeq.) ggtggtcacgaaggtgctggtgtcgttgtcggcatgggtgaaaacgtcaagggctggaagattggtgacttcgccggtatcaaatggttgaacggttcctgtatgtcctgtgagttctgtcaacaaggtgctgaaccaaactgtggtgaggccgacttgtctggttacacccacgatggttctttcgaacaatacgccactgctgatgctgttcaagccgccagaatcccagctggtactgatttggccgaagttgccccaatcttgtgtgcgggtgtcaccgtctacaaagccttgaagactgccgacttggccgctggtcaatgggtcgctatctccggtgctggtggtggtttgggttccttggctgtccaatacgccgtcgccatgggcttgagagtcgttgccattgacggtggtgacgaaaagggtgcctttgtcaagtccttgggtgctgaagcctacattgatttcctcaaggaaaaggacattgtctctgctgtcaagaaggccaccgatggaggtccacacggtgctatcaatgtttccgtttccgaaaaagccattgaccaatccgtcgagtacgttagaccattgggtaaggttgttttggttggtttgccagctggctccaaggtcactgctggtgttttcgaagccgttgtcaagtccattgaaatcaagggttcctatgtcggtaacagaaaggataccgccgaagccgttgactttttctccagaggcttgatcaagtgtccaatcaagattgttggcttgagtgaattgccacaggtcttcaagttgatggaagaaggtaagatcttgggtagatacgtcttggatacctccaaa SEQ ID ADH1-2 short,MSANIPKTQKAVVFEKNGGELKYKDIPVPTPKANELLINVKYSGVCHT NO: 154 CandidaDLHAWKGDWPLDTKLPLVGGHEGAGVVVGMGENVKGWKIGDFAGI (Amino AcidKWLNGSCMSCEFCQQGAEPNCGEADLSGYTHDGSFEQYATADAVQ Seq.)AARIPAGTDLAEVAPILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYAVAMGLRVVAIDGGDEKGDFVKSLGAEAYIDFLKEKGIVAAVKKATDGGPHGAINVSVSEKAIDQSVEYVRPLGKVVLVGLPAGSKVTAGVFEAVVKSIEIKGSYVGNRKDTAEAVDFFSRGLIKCPIKIVGLSELPQ VFKLMEEGKILGRYVLDTSKSEQ ID ADH1-2 short,atgcatgcattattctcaaaatcagtttttctcaagtatgtgagtctgcccactacctctgctatcccccaNO: 155 Candidattccctagaattcattgtctcccgaagctcctatttaaggagacgaattcccccatatcttccacgttgc(Nucleic AcidtcccactttccttccttctattattcttcttcttcagtctacaccaagaaatcatttcacacaatgtctgctaSeq.) atatcccaaaaactcaaaaagctgtcgtcttcgagaagaacggtggtgaattaaaatacaaagacatcccagtgccaaccccaaaggccaacgaattgctcatcaacgtcaagtactcgggtgtctgtcacactgatttgcacgcctggaagggtgactggccattggacaccaaattgccattggttggtggtcacgaaggtgctggtgttgttgtcggcatgggtgaaaacgtcaagggctggaaaatcggtgatttcgccggtatcaaatggttgaacggttcttgtatgtcctgtgagttctgtcagcaaggtgctgaaccaaactgtggtgaagctgacttgtctggttacacccacgatggttctttcgaacaatacgccactgctgatgctgtgcaagccgccagaatcccagctggcactgatttggccgaagttgccccaatcttgtgtgctggtgtcaccgtctacaaagccttgaagactgccgacttggctgctggtcaatgggtcgctatctccggtgctggtggtggtttgggctccttggctgtccaatacgccgtcgccatgggtttgagagtcgttgccattgacggtggtgacgaaaagggtgactttgtcaagtccttgggtgctgaagcctacattgatttcctcaaggaaaagggcattgttgctgctgtcaagaaggccactgatggcggtccacacggtgctatcaatgtttccgtttccgaaaaagccattgaccaatctgtcgagtacgttagaccattgggtaaggttgttttggttggtttgccagctggctccaaggtcactgctggtgttttcgaagccgttgtcaagtccattgaaatcaagggttcttacgtcggtaacagaaaggatactgccgaagccgttgactttttctccagaggcttgatcaagtgtccaatcaagattgtgggcttgagtgaattgccacaggtcttcaagttgatggaagaaggtaagatcttgggtagatacgtcttggatacctccaaa SEQ ID MAE1, ScMWPIQQSRLYSSNTRSHKATTTRENTFQKPYSDEEVTKTPVGSRAR NO: 156 (Amino AcidKIFEAPHPHATRLTVEGAIECPLESFQLLNSPLFNKGSAFTQEEREAF Seq.) in Spec.NLEALLPPQVNTLDEQLERSYKQLCYLKTPLAKNDFMTSLRVQNKVLYFALIRRHIKELVPIIYTPTEGDAIAAYSHRFRKPEGVFLDITEPDSIECRLATYGGDKDVDYIVVSDSEGILGIGDQGIGGVRIAISKLALMTLCGGIHPGRVLPVCLDVGTNNKKLARDELYMGNKFSRIRGKQYDDFLEKFIKAVKKVYPSAVLHFEDFGVKNARRLLEKYRYELPSFNDDIQGTGAVVMASLIAALKHTNRDLKDTRVLIYGAGSAGLGIADQIVNHMVTHGVDKEEARKKIFLMDRRGLILQSYEANSTPAQHVYAKSDAEWAGINTRSLHDVVENVKPTCLVGCSTQAGAFTQDVVEEMHKHNPRPIIFPLSNPTRLHEAVPADLMKWTNNNALVATGSPFPPVDGYRISENNNCYSFPGIGLGAVLSRATTITDKMISAAVDQLAELSPLREGDSRPGLLPGLDTITNTSARLATAVILQALEEGTARIEQEQVPGGAPGETVKVPRDFDECLQWVKAQMWE PVYRPMIKVQHDPSVHTNQL*SEQ ID ZWF1, MSFDPFGSTATIVVFGASGDLAKKKTFPALFGLFREGHLSSDVKIIGYA NO: 157Scheffersomyces RSHLEEDDFKKRISANFKGGNPETVEQFLKLTSYISGPYDTDEGYQTLstipitis LKSIEDYEAANNVSTPERLFYLALPPSVFTTVASQLKKNVYSETGKTRI (Amino AcidIVEKPFGHDLESSRQLQKDLSPLFTEEELYRIDHYLGKEMVKNLLVLR Seq.)FGNELFNGVWNKNHIKSIQISFKEAFGTDGRGGYFDSIGIVRDVMQNHLLQVLTLLTMDRPVSFDPEAVRDEKVKILKAFDALDPEDILLGQYGKSEDGSKPGYLDDSTVPKDSKCandidaTYAALGIKIHNERWEGVPIVMRAGKALDESKVEIRIQFKPVARGMFKEIQRNELVIRVQPNESIYLKINSKIPGISTETSLTDLDLTYSTRYSKDFWIPEAYEALIRDCYLGNHSNFVRDDELDVSWKLFTPLLQYIESDKSPQPEVYAYGSKGPKGLREFLNKHDYIFAD EGTYQWPLTTPKVKGKI*SEQ ID ACS2B, MPALFKDSAQHILDTIKSELPLDPLKTAYAVPLENSAEPGYSAIYRNKY NO: 158Candida STDKLIDTPYPGLDTLYKLFEVSTEANGDKPCLGGRVKNADGTFGEY (Amino AcidKFQDYNTIHQRRNNLGSGIFFVLQNNPYKTNSEAHSKLKYDPTSKDS Seq.)(fromFILTIFSHNRPEWALCDLTSIAYSITNTALYDTLGPDTSKYILGLTESPIV spec)VCSKDKIRGLIDLKKNNPDELSNLIVLVSMDDLTTADASLKNYGSEHNVTVYDIKQVEKLGEINPLDPIEPTPDTNFTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGQFNPTAYSFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGLALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQMKEENENFNPSFIVYDRLLNLLRKKVGLQKVSQISTGSAPISPSTIQFLKASLNVGILQGYGLSESFAGCMASSKFEPAAVTCGPPGITTEVKLKDLEEMGYTSKDEGGPRGELLLRGPQIFKEYFKNPEETAEAIDEDGWFHTGDVAKINNKGRISIIDRAKNFFKLAQGEYVTPEKIEGLYLSKFPYIAQIFVHGDSKESYLVGVVGLDPVAGKQYMESRFHDKIIKEEDVVEFFKSPRNRKILLQDMNKSIADQLQGFEKLHNIYVDFDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRNGSL* SEQ ID ACS2C,MPALFKDSAKHIFDTIKSELPLDPLKTAYAVPLENSAEPGYSAIYRNKY NO: 159 CandidaSIDKLIDTPYPGLDTLYKLFEVATEAYGDKPCLGARVKNADGTFGEYK (Amino AcidFQDYNTIHQRRNNFGSGIFFVLQNNPYKTDSEAHSKLKYDPTSKDSFI Seq.)(fromLTIFSHNRPEWALCDLTSIAYSITNTALYDTLGPDTSKYILGLTESPIVIC spec)SKDKIRGLIDLKKNNPDELSNLIVLVSMDDLTTADASLKNYGSEHNVTVFDIKQVEKLGEINPLDPIEPTPDTNFTITFTSGTTGANPKGVLLNHRNAVAGVTFVLSRYDGHFNPTAYSFLPLAHIYERASIQFALTIGSAIGFPQGPSPLTLIEDAKVLQPDGLALVPRVLTKLEAAIRAQTVNNDEKPLVKSVFGAAINAKMEAQMKEENENFNPSFIVYDRLLNLLRKKVGLQKVTQISTGSAPISPSTIQFLKASLNVGILQGYGLSESFAGCMASSKFEPAAATCGPTGVTTEVKLKDLEEMGYTSKDEGGPRGELLLRGPQIFKGYFKNPEETAKAIDEDGWFHTGDVAKINDKGRISIIDRAKNFFKLAQGEYVTPEKIEGLYLSKFPYIAQLFVHGDSKESYLVGVVGLDPVAGKQYMESRFHDKIIKEEDVVEFFKSPRNRKILVQDMNKSIADQLQGFEKLHNIYVDFDPLTVERGVITPTMKIRRPLAAKFFQDQIDAMYSEGSLVRNGSL* SEQ ID PAA073aaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgNO: 160 (Nucleic AcidcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagcSeq.) cgaacgaccgagcgcagcgagtcagtgagcgaggaagcgagttcggcatggcagatcatcatgcctgcaggagctccaattgtaatatttcgggagaaatatcgttggggtaaaacaacagagagagagagggagagatggttctggtagaattataatctggttgttgcaaatgctactgatcgactctggcaatgtctgtagctcgctagttgtatgcaacttaggtgttatgcatacacacggttattcggttgaattgtggagtaaaaattgtctgagttgtgtcttagctactggctggccccccgcgaaagataatcaaaattacacttgtgaatttttgcacacacaccgattaacatttcccttttttgtccaccgatacacgcttgcctcttcttttttttctctgtgcttccccctcctgtgactttttccaccattgatataaaatcaactccatttccctaaaatctccccagattctaaaaacaacttcttctcttctgcttttcctttttttttgttatatttatttaccatcccttttttttgaatagttattccccactaacattgttcaaatcttcacgacataagaagagcccgggtctagatgtgtgctcttccgagtgactcttttgataagagtcgcaaatttgatttcataagtatatattcattatgtaaagtagtaaatggaaaattcattaaaaaaaaagcaaatttccgttgtatgcatactccgaacacaaaactagccccggaaaaacccttagttgatagttgcgaatttaggtcgaccatatgcgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcaatggttagcacaaaaacatacacagaaagggcatcagcacacccctccaaggttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgaccacaaccgccgagttcctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatcgatatcatctcagacttcagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgggttcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggggtataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgttgaagggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatgttggcggagttgtcgagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatcgggagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaagggtttgattggatcatcatgacgcctggtgtggggttggatgataaaggcgatgcgttgggccagcagtataggactgttgatgaggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaaaggaagagaccctgaggtggagggaaagagatacagggatgctggatggaaggcatacttgaagagaactggtcagttagaataaatattgtaataaataggtctatatacatacactaagcttctaggacgtcattgtagtcttcgaagttgtctgctagtttagttctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgcgttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcacttgcttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtctacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaa SEQ ID pAA298gatctggaatccctcggcgtcggtcttgggggtgggggcattctttcttggtcttgggaacgccaacgNO: 161 (Nucleic AcidctttgttgtttgggttcttgaacacggactgctcgaaaaagtaccagtatgatgccttacctctgctggcSeq.) tttccacaagtatgggagggcattgacagcgactgtcttggctaacagcacgtcgtcggcaattaaatatttggcttccaataactgactaccaaggatggcagcagcggctatttctaatcctgacatgtttctcgtacgtagtagtgaatgaagggaaggtggaataatatcaagggcgaattctgcagatatccatcacactggcggccgctcgagcatgcatctagagggcccaattcgccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctatacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgccggggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttttcacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgaggagggccaccatggccaagttgaccagtgccgttccggtgctcaccgcgcgcgacgtcgccggagcggtcgagttctggaccgaccggctcgggttctcccgggacttcgtggaggacgacttcgccggtgtggtccgggacgacgtgaccctgttcatcagcgcggtccaggaccaggtggtgccggacaacaccctggcctgggtgtgggtgcgcggcctggacgagctgtacgccgagtggtcggaggtcgtgtccacgaacttccgggacgcctccgggccggccatgaccgagatcggcgagcagccgtgggggcgggagttcgccctgcgcgacccggccggcaactgcgtgcacttcgtggccgaggagcaggactgacacgtgctaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgggcttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctatttaggtgacactatagaatactcaagctatgcatcaagcttggtaccgagctcggatccactagtaacggccgccagtgtgctggaattcgcccttccgttaaacaaaaatcagtctgtaaaaaaggttctaaataaatattctgtctagtgtacacattctcccaaaatagtgaaatccagctctacaatttggctttaccggtacaaatcaaagaccaatcgtcttcagtcaactcctggtacttgtcgccattcaaccagtagatcaaatccttgccgtcttcaccctttggcaacttttggttcttgaattggttcttaggaaccttgtggttgtgcgaagcctcaatggtgccaatcttgatgaacgcaggttgagcatacacaggcaaagacttggtcacgtgagagtgaatcaatttcaagatttcttcatggctcaactcgtccttggcttcacagacggcaaaacaggctctaccttcgtggtttggcaccttgacaccgacaacgacggactgcttcaaggccttggagcccatcaattcgttctcgacctcggtggcggagacgttttcggacttccaacggaaagtgtcacctaatctgtcgacaaagtacaacaatttgtcctcgtccatcttcaacaagtcaccggatctgtaccacgcgtcaccttttttgaaaacattggtgaggattttgctgttggtggcggacttgttaccataataaccctggaaggatttctgcacgtcgttagggttcaagattctcatcaacaactcacctggctcgttgtaagcggcctcggtacagaacccggtcttggggtccttgtagatttcactctcgtcttctgggtccatcttggccaatttctgctgggtagacaataacaagctgatgagggacccgtacttacgacaggcgccgacaccgtactcaccgtactgcaagttggtggtggcgataggggactcggtggcggcgtagaactcaccgataccttcaatgtggaatctgcgcttgaactcagaccatatatctggacgcaacccgttaccgtaggcaattctgacattgtgtctgtcttggtctggatgaggcttggagttcaacaagtaacgacagacctcaccgacgtattgcacgtgggtggcaccacataatctggcctgggtccagaacgaagtagcggagaatttctgggacacggagacacagccaccgacaatcaaagtaggacacaaccccaacatggccgcggtggagtggtacaagggcatggcggtcaagacgttcgatttcgagtcaatcttcatgatgtggccaaagaaaaccgaggccatgaaggcttttctccaggacatgataccggcttttggcaaaccggtggtacccgaggtgtaaatcaatgcacaagcggaggagtcagtatcggttggtcttctggtcttgtcctcggctctgtgttttggagtcgacttgagtctcaatctgtcaaacaaggcaaactcgtcaatgtagtttatttgcacatgtggcaattcctctctgatctgagcctcggtatctctgattggggaatcacagtccgggtcaacgaaaacttgcgaagcgttgacaatcttaagacagtggatcaatggcttgtccttggtgttgaagttcaagaacgcaggcaaggcaccaatgttccacaatgccaaccacaagacaatgaaaagcggcttgttcatacaagaaacaccgatggtgtcgttggcagtgacgccgtactcgttcttcaagatgtatgagtacttcaaaaccatgtcgtacaattccttgtaggtgtattcttctaggtcaaactgatcgtcgtagatccactagtaacggccgccagtgtgctggaattcgcccttgggctaacgaaaaggaaaccgctgacgttaaaggtatctacggttgtttcggtatgacccccggggatctgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcaatggttagcacaaaaacatacacagaaagggcatcagcacacccctccnaggttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgaccacaaccgccgagttcctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatcgatntcatctcagacttcagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgggttcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggggtataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgttgaagggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatgtnggcggagttgtcgagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatcgggagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaagggtttgattggatcatcatgacgcctggtgtggggttggatgataaaggcgatgcgttgggccagcagtataggactgttgatgaggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaaaggaagagaccctgaggtggagggaaagagatacagggatgctggatggaaggcatacttgaagagaactggtcagttagaataaatattgtaataaataggtctatatacatacactaagcttctaggacgtcattgtagtcttcgaagttgtctgctagtttagttctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgcgttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcacttgcttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtctacggtcgacgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaagttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatgagatttatccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagcaaacgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcagagctcggtacccggg SEQ ID >Piece A (5′gtttgtctatgttttccgtttgccttttctttctagtacgagacgttattgaacgaagtttttatatatctagatcNO: 162 untranslatedtaatacatattccatgtctgttcatttttgacggagtttcataaggtggcagtttctaatcaaaggtccgtregion ofcattggcgtcgtggcattggcggctcgcatcaactcgtatgtcaatattttctgttaactccgccagacFAO1 (from atacgatcaaaacctacaagcaaaaaaattccac position −500 to −250)(Nucleic Acid Seq.) SEQ ID >Piece B -cgacgggtacaacgagaattgtattgaattgatcaagaacatgatcttggtgttacagaacatcaaNO: 163 URA3 markergttcttggaccagactgagaatgcacagatatacaaggcgtcatgtgataaaatggatgagattta(Nucleic Acidtccacaattgaagaaagagtttatggaaagtggtcaaccagaagctaaacaggaagaagcaa Seq.)acgaagaggtgaaacaagaagaagaaggtaaataagtattttgtattatataacaaacaaagtaaggaatacagatttatacaataaattgccatactagtcacgtgagatatctcatccattccccaactcccaagaaaaaaaaaaagtgaaaaaaaaaatcaaacccaaagatcaacctccccatcatcatcgtcatcaaacccccagctcaattcgcaatggttagcacaaaaacatacacagaaagggcatcagcacacccctccaaggttgcccaacgtttattccgcttaatggagtccaaaaagaccaacctctgcgcctcgatcgacgtgaccacaaccgccgagttcctttcgctcatcgacaagctcggtccccacatctgtctcgtgaagacgcacatcgatatcatctcagacttcagctacgagggcacgattgagccgttgcttgtgcttgcagagcgccacgggttcttgatattcgaggacaggaagtttgctgatatcggaaacaccgtgatgttgcagtacacctcgggggtataccggatcgcggcgtggagtgacatcacgaacgcgcacggagtgactgggaagggcgtcgttgaagggttgaaacgcggtgcggagggggtagaaaaggaaaggggcgtgttgatgttggcggagttgtcgagtaaaggctcgttggcgcatggtgaatatacccgtgagacgatcgagattgcgaagagtgatcgggagttcgtgattgggttcatcgcgcagcgggacatggggggtagagaagaagggtttgattggatcatcatgacgcctggtgtggggttggatgataaaggcgatgcgttgggccagcagtataggactgttgatgaggtggttctgactggtaccgatgtgattattgtcgggagagggttgtttggaaaaggaagagaccctgaggtggagggaaagagatacagggatgctggatggaaggcatacttgaagagaactggtcagttagaataaatattgtaataaataggtctatatacatacactaagcttctaggacgtcattgtagtcttcgaagttgtctgctagtttagttctcatgatttcgaaaaccaataacgcaatggatgtagcagggatggtggttagtgcgttcctgacaaacccagagtacgccgcctcaaaccacgtcacattcgccctttgcttcatccgcatcacttgcttgaaggtatccacgtacgagttgtaatacaccttgaagaacggcttcgtct SEQ ID >Piece CGttaactccgccagacatacgatcaaaacctacaagcaaaaaaattccac NO: 164 (Nucleic AcidSeq.) SEQ ID >Piece D -Gagctccaattgtaatatttcgggagaaatatcgttggggtaaaacaacagagagagagaggg NO: 165PromoteragagatggttctggtagaattataatctggttgttgcaaatgctactgatcgactctggcaatgtctgtaPDX4gctcgctagttgtatgcaacttaggtgttatgcatacacacggttattcggttgaattgtggagtaaaa(Nucleic AcidattgtctgagttgtgtcttagctactggctggccccccgcgaaagataatcaaaattacacttgtgaatSeq.)ttttgcacacacaccgattaacatttcccttttttgtccaccgatacacgcttgcctcttcttttttttctctgtgcttccccctcctgtgactttttccaccattgatataaaatcaactccatttccctaaaatctccccagattctaaaaacaacttcttctcttctgcttttcctttttttttgttatatttatttaccatcccttttttttgaatagttattccccactaacattgttcaaatcttcacgacata SEQ ID >Piece D -Gaagatgaagcgtatgagtattatgagtactgtcggacgttggaaggtggcagagttaagcccg NO: 166Promoter agaaagcaaggaaggagtgggagatgatgagtgatgcggccaaagaggatgtgaaggctgcPEX11gtatctgtttttgatagctggtggtagccgaatagaggaaggcaagcttgttcatattggatgatgatg(Nucleic AcidgtagatggtggctgccaaagtggttgtaaatagaaaaaagtgggtttgggtctgttgatagttagtgSeq.) gtggcggctgtctgtgattacgtcagcaagtagcacctcggcagttaaaacagcagcaacagaaaaaaaatgtgtgaaagtttgattcccccacagtctaccacacccagagttccatttatccataatatcacaagcaatagaaaaataaaaaattatcaacaaatcacaacgaaaagattctgcaaaattattttcacttcttcttttgacttcctcttcttcttgttaggttctttccatattttccccttaaacccatacacaacgcagcc SEQ ID >Piece D -Ctagcaaaggcttgatcagagaaagcaacaaaaaaaaaaactctaatactccagaatacactc NO: 167PromoterctttagaaacacacaacaaacaagcctagactaccatggactacgatgaagacgatttagattacTEF1 (NucleicatttctcaaggagaagaggaagagtttgacgaaaacaagttgaacaacgaagagtacgacttgtAcid Seq.)tgcatgacatgcttccggagttgaagacaaaattgaaagattacaatgatgagatcccagattacgatttaaaggaagcgttatactacaactatttcgagatagaccctaccattgaagaattgaagacgaaattcaaaaagagtacgtatatacaactaacatcaacgcctttctagtttctgttctgtctccaatgcttctcctggtttcttcatggttctctctgtaccaacaaggaaaaaaaaaaaaatctggcaaaaaaaaccaaaccaaccaatgttcttactcaccaacgccctacaatc SEQ ID >Piece E-atggctccatttttgcccgaccaggtcgactacaaacacgtcgacacccttatgttattatgtgacggNO: 168 First 250 bp ofgatcatccacgaaaccaccgtggacgaaatcaaagacgtcattgcccctgacttccccgccgacthe codingaaatacgaggagtacgtcaggacattcaccaaaccctccgaaaccccagggttcagggaaaccsequence of gtctacaacaccgtcaacgcaaacaccatggatgcaatccaccagttcattatctFAO1. (Nucleic Acid Seq.) SEQ ID >PGKpromoter_ttgtccaatgtaataatttttccatgactaaaaagtgtgtgttggtgtaaagaagaaagtggaagggaNO: 169 Candida_cgttggtgatggtgagttcgtctatcccttttttatagttgcttgtatagtaggctactcttctagggactcgATCC20336atgggggaaggttcttgatatttgcttagttcgagaaggttccagatgagcgagacatttttggtacgacattgggtggatgatctgcacgacattttgtgattcttgcgacacgctgcactaccaagtgtagtctggctgaacggatcacaagataaacctctgaaaaattatctcagggcatgcaacaacaattatacatagaagagggagtcacgatatacacctgtgaaggaatcatgtggtcggctctccttgaactttgaattcatgcaattattaagaagaagcacaggtgagcaacccaccatacgttcatttgcaccacctgatgattaaaagccaaagaaagaaaaaaaaaaaagaaacaggcggtgggaattgttacaacccacgcgaacccgaaaatggagcaatcttccccggggcctccaaataccaactcacccgagagagagaaagagacaccacccaccacgagacggagtatatccaccaaggtaagtaactcagggttaatgatacaggtgtacacagctccttccctagccattgagtgggtatcacatgacactggcaggttacaaccacgtttagtagttattttgtgcaattccatggggatcaggaagtttggtttggtgggtgcgtctactgattcccctttgtctctgaaaatcttttccctagtggaacactttggctgaatgatataaattcaccttgattcccaccctcccttctttctctctctctctgttacacccaattgaattttcttttttttttttttactttccctccttctttatcatcaaagataagtaagtttatcaattgcctattcaga

Example 58: Examples of Certain Non-Limiting Embodiments

-   A1. A genetically modified yeast, comprising:    -   one or more genetic modifications that substantially block beta        oxidation activity; and    -   one or more genetic modifications that increase one or more        activities chosen from monooxygenase activity, monooxygenase        reductase activity, thioesterase activity, acyltransferase        activity, isocitrate dehydrogenase activity,        glyceraldehyde-3-phosphate dehydrogenase activity,        glucose-6-phosphate dehydrogenase activity, acyl-coA oxidase        activity, fatty alcohol oxidase activity, acyl-CoA hydrolase        activity, alcohol dehydrogenase activity, peroxisomal biogenesis        factor activity, and fatty aldehyde dehydrogenase activity.-   A2. The genetically modified yeast of embodiment A1, wherein the one    or more genetic modifications increase one or more monooxygase    activities chosen from a CYP52A12 monooxygenase activity, CYP52A13    monooxygenase activity, CYP52A14 monooxygenase activity, CYP52A15    monooxygenase activity, CYP52A16 monooxygenase activity, CYP52A17    monooxygenase activity, CYP52A18 monooxygenase activity, CYP52A19    monooxygenase activity, CYP52A20 monooxygenase activity, CYP52D2    monooxygenase activity and BM3 monooxygenase activity.-   A3. The genetically modified yeast of embodiment A1 or A2, wherein    the one or more genetic modifications increase one or more    monooxygenase reductase activities chosen from CPRA monooxygenase    reductase activity, CPRB monooxygenase reductase activity and CPR750    monooxygenase reductase activity.-   A4. The genetically modified yeast of any one of embodiments A1 to    A3, wherein the one or more genetic modifications increase a IDP2    isocitrate dehydrogenase activity.-   A5. The genetically modified yeast of any one of embodiments A1 to    A4, wherein the one or more genetic modifications increase a GDP1    glyceraldehyde-3-phosphate dehydrogenase activity.-   A6. The genetically modified yeast of any one of embodiments A1 to    A5, wherein the one or more genetic modifications increase one or    more glucose-6-phosphate dehydrogenase activities chosen from a ZWF1    glucose-6-phosphate dehydrogenase activity and ZWF2    glucose-6-phosphate dehydrogenase activity.-   A7, The genetically modified yeast of any one of embodiments A1 to    A6, wherein the one or more genetic modifications increase one or    more fatty alcohol oxidase activities chosen from FAO1 fatty alcohol    oxidase activity, FAO2A fatty alcohol oxidase activity, FAO2B fatty    alcohol oxidase activity, FAO13 fatty alcohol oxidase activity,    FAO17 fatty alcohol oxidase activity, FAO18 fatty alcohol oxidase    activity and FAO20 fatty alcohol oxidase activity.-   A8. The genetically modified yeast of any one of embodiments A1 to    A7, wherein the one or more genetic modifications increase one or    more alcohol dehydrogenase activities chosen from ADH1 alcohol    dehydrogenase activity, ADH2 alcohol dehydrogenase activity, ADH3    alcohol dehydrogenase activity, ADH4 alcohol dehydrogenase activity,    ADH5 alcohol dehydrogenase activity, ADH7 alcohol dehydrogenase    activity, ADH8 alcohol dehydrogenase activity and SFA alcohol    dehydrogenase activity.-   A9. The genetically modified yeast of any one of embodiments A1 to    A8, wherein the one or more genetic modifications increase one or    more acyl-CoA hydrolase activities chosen from ACH-A acyl-CoA    hydrolase activity and ACH-B acyl-CoA hydrolase activity.-   A10. The genetically modified yeast of any one of embodiments A1 to    A9, wherein the one or more genetic modifications increase one or    more acyltransferase activities chosen from acyl-CoA sterol    acyltransferase activity, diacylglycerol acyltransferase activity    and phospholipid:diacylglycerol acyltransferase activity.-   A11. The genetically modified yeast of embodiment A10, wherein the    one or more acyltransferase activities are chosen from ARE1 acyl-CoA    sterol acyltransferase activity, ARE2 acyl-CoA sterol    acyltransferase activity, DGA1 diacylglycerol acyltransferase    activity, and LRO1 phospholipid:diacylglycerol acyltransferase    activity.-   A12. The genetically modified yeast of any one of embodiments A1 to    A11, wherein the one or more genetic modifications increase an    acyl-coA thioesterase activity.-   A13. The genetically modified yeast of embodiment A12, wherein the    acyl-coA thioesterase activity is a TESA acyl-coA thioesterase    activity.-   A14. The genetically modified yeast of any one of embodiments A1 to    A13, wherein the one or more genetic modifications increase a PEX11    peroxisomal biogenesis factor activity.-   A15. The genetically modified yeast of any one of embodiments A1 to    A14, wherein the one or more genetic modifications increase one or    more fatty aldehyde dehydrogenase activities chosen from HFD1 fatty    aldehyde dehydrogenase activity and HFD2 fatty aldehyde    dehydrogenase activity.-   A16. The genetically modified yeast of any one of embodiments A1 to    A15, wherein the one or more genetic modifications increase a POX5    acyl-coA oxidase activity.-   A17. The genetically modified yeast of any one of embodiments A1 to    A16, wherein the one or more genetic modifications increase a    monooxygenase activity and a monooxygenase reductase activity.-   A18. The genetically modified yeast of embodiment A17, wherein the    one or more genetic modifications increase a CYP52A19 monooxygenase    activity and a CPRB monooxygenase reductase activity.-   A19. The genetically modified yeast of embodiment A17, wherein the    one or more genetic modifications increase a CYP52A14 monooxygenase    activity and a CPRB monooxygenase reductase activity.-   A20. The genetically modified yeast of any one of embodiments A1 to    A19, wherein the one or more genetic modifications increase a    monooxygenase activity, a monooxygenase reductase activity, and a    isocitrate dehydrogenase activity.-   A21. The genetically modified yeast of embodiment A20, wherein the    one or more genetic modifications increase a CYP52A19 monooxygenase    activity, a CPRB monooxygenase reductase activity, and a IDP2    isocitrate dehydrogenase activity.-   A22. The genetically modified yeast of any one of embodiments A1 to    A21, wherein the one or more genetic modifications increase a    monooxygenase activity, a monooxygenase reductase activity, and a    glucose-6-phosphate dehydrogenase activity.-   A23. The genetically modified yeast of embodiment A22, wherein the    one or more genetic modifications increase a CYP52A19 monooxygenase    activity, a CPRB monooxygenase reductase activity, and a ZWF1    glucose-6-phosphate dehydrogenase activity.-   A24. The genetically modified yeast of any one of embodiments A1 to    A23, wherein a monooxygenase activity is by a polypeptide comprising    an amino acid sequence chosen from SEQ ID NOs: 52, 53, 54, 55, 56,    57, 58, 59, 60 and 61.-   A25, The genetically modified yeast of embodiment A24, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24.-   A26. The genetically modified yeast of any one of embodiments A1 to    A25, wherein a monooxygenase reductase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 28, 63, 64    and 65.-   A27. The genetically modified yeast of embodiment A26, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    24, 25, 26 and 27.-   A28. The genetically modified yeast of any one of embodiments A1 to    A27, wherein a thioesterase activity is by a polypeptide comprising    an amino acid sequence of SEQ ID NO: 38.-   A29. The genetically modified yeast of embodiment A28, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 37.-   A30. The genetically modified yeast of any one of embodiments A1 to    A29, wherein an acyltransferase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 44, 46, 48    and 50.-   A31. The genetically modified yeast of embodiment A30, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    43, 45, 47 and 49.-   A32. The genetically modified yeast of any one of embodiments A1 to    A31, wherein an isocitrate dehydrogenase activity is by a    polypeptide comprising an amino acid sequence of SEQ ID NO: 67, 69    or 100.-   A33. The genetically modified yeast of embodiment A32, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 68, 70 or    99.-   A34. The genetically modified yeast of any one of embodiments A1 to    A33, wherein a glyceraldehyde-3-phosphate dehydrogenase activity is    by a polypeptide comprising an amino acid sequence of SEQ ID NO: 72.-   A35. The genetically modified yeast of embodiment A34, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 71.-   A36. The genetically modified yeast of any one of embodiments A1 to    A35, wherein a glucose-6-phosphate dehydrogenase activity is by a    polypeptide comprising an amino acid sequence of SEQ ID NO: 74, 76    or 157.-   A37. The genetically modified yeast of embodiment A36, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 73 or 75.-   A38, The genetically modified yeast of any one of embodiments A1 to    A37, wherein an acyl-coA oxidase activity is by a polypeptide    comprising an amino acid sequence of SEQ ID NO: 32.-   A39. The genetically modified yeast of embodiment A38, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 31.-   A40. The genetically modified yeast of any one of embodiments A1 to    A39, wherein a fatty alcohol oxidase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 3, 5, 7,    9, 11, 13, 132 and 134.-   A41. The genetically modified yeast of embodiment A40, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    2, 4, 6, 8, 10, 12, 131 and 133.-   A42. The genetically modified yeast of any one of embodiments A1 to    A41, wherein an acyl-CoA hydrolase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 34 and 36.-   A43. The genetically modified yeast of embodiment A42, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    33 and 35.-   A44. The genetically modified yeast of any one of embodiments A1 to    A43, wherein an alcohol dehydrogenase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 129, 113,    115, 117, 119, 121, 123, 125, 127, 152 and 154.-   A45. The genetically modified yeast of embodiment A44, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    130, 114, 116, 118, 120, 122, 124, 126, 128, 153 and 155.-   A46. The genetically modified yeast of any one of embodiments A1 to    A45, wherein a peroxisomal biogenesis factor activity is by a    polypeptide comprising an amino acid sequence of SEQ ID NO: 96.-   A47. The genetically modified yeast of embodiment A46, wherein the    polypeptide is encoded by a polynucleotide of SEQ ID NO: 95.-   A48. The genetically modified yeast of any one of embodiments A1 to    A47, wherein a fatty aldehyde dehydrogenase activity is by a    polypeptide comprising an amino acid sequence chosen from SEQ ID    NOs: 139 and 141.-   A49. The genetically modified yeast of embodiment A48, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    140 and 142.-   A50, The genetically modified yeast of any one of embodiments A1 to    A49, comprising one or more genetic modifications that decrease an    acyl-coA synthetase activity.-   A51. The genetically modified yeast of embodiment A50, wherein the    one or more genetic modifications decrease one or more acyl-coA    synthetase activities chosen from ACS1 acyl-coA synthetase activity    and FAT1 long-chain acyl-CoA synthetase activity.-   A52. The genetically modified yeast of embodiment A50 or A51,    wherein the one or more genetic modifications disrupt a nucleic acid    that encodes a polypeptide having the acyl-coA synthetase activity.-   A53. The genetically modified yeast of any one of embodiments A50 to    A52, wherein the acyl-coA synthetase activity is by a polypeptide    comprising an amino acid sequence chosen from SEQ ID NOs: 40, 42,    80, 82, 84, 90, 158 and 159.-   A54. The genetically modified yeast of embodiment A53, wherein the    polypeptide is encoded by a polynucleotide chosen from SEQ ID NOs:    39, 41, 79, 81, 83 and 89.-   A55. The genetically modified yeast of any one of embodiments A1 to    A54, which is a Candida spp. yeast.-   A56. The genetically modified yeast of embodiment A55, wherein the    Candida spp. yeast is chosen from C. tropicalis and C. viswanathii.-   A57. The genetically modified yeast of embodiment A56, wherein the    Candida spp. yeast is a genetically modified ATCC20336 yeast.-   A58. The genetically modified yeast of any one of embodiments A1 to    A57, which is chosen from a Yarrowia spp. yeast, Pichia spp. yeast,    Saccharomyces spp. yeast and Kluyveromyces spp. yeast.-   A59. The genetically modified yeast of embodiment A58, which is    chosen from Y. lipolytica, P. pastoris, P. membranifaciens, P.    kluyveri, P. guilliermondii, P. heedii, P. subpelliculosa, S.    cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis, K. lactis    and K. marxianus.-   A60. The genetically modified yeast of any one of embodiments A1 to    A59, which is capable of producing a diacid from a feedstock    comprising one or more components from a vegetable oil.-   A61. The genetically modified yeast of embodiment A60, wherein the    diacid is a C4 to C24 diacid.-   A62. The genetically modified yeast of embodiment A61, wherein the    diacid is a 010, C12, C14, C16, C18 or C20 diacid.-   A62.1. The genetically modified yeast of embodiment A62, wherein the    diacid is a 010 diacid.-   A63. The genetically modified yeast of embodiment A62, wherein the    diacid is a C12 diacid.-   A64. The genetically modified yeast of embodiment A62, wherein the    diacid is a C18 diacid.-   A65. The genetically modified yeast of any one of embodiments A60 to    A64, wherein the diacid contains no unsaturation.-   A66. The genetically modified yeast of any one of embodiments A60 to    A64, wherein the diacid contains one or more unsaturations.-   A67. The genetically modified yeast of any one of embodiments A60 to    A66, wherein the diacid is the predominant diacid in a mixture of    diacids.-   A68. The genetically modified yeast of any one of embodiments A60 to    A67, wherein the feedstock comprises a substantially pure oil.-   A69. The genetically modified yeast of any one of embodiments A60 to    A68, wherein the feedstock comprises a plurality of fatty acids.-   A70. The genetically modified yeast of embodiment A69, wherein the    feedstock comprises a soapstock.-   A71. The genetically modified yeast of embodiment A69, wherein the    feedstock comprises a fatty acid distillate.-   A72. The genetically modified yeast of any one of embodiments A60 to    A71, wherein the vegetable oil is from a plant chosen from palm,    palm kernel, coconut, soy, safflower, canola or combination thereof.-   A73. The genetically modified yeast of any one of embodiments A1 to    A73, wherein a genetic modification that increases an activity    comprises incorporating in the yeast multiple copies of a    polynucleotide that encodes a polypeptide having the activity.-   A74. The genetically modified yeast of any one of embodiments A1 to    A73, wherein a genetic modification that increases an activity    comprises incorporating in the yeast a promoter in operable linkage    with a polynucleotide that encodes a polypeptide having the    activity.-   A75. The genetically modified yeast of embodiment A74, wherein the    promoter is native to the yeast.-   A76. The genetically modified yeast of embodiment A74 or A75,    wherein the promoter is chosen from a POX4 promoter, PEX11 promoter,    TEF1 promoter, PGK promoter and FAO1 promoter.-   A77. The genetically modified yeast of embodiment A76, wherein the    promoter comprises a polynucleotide chosen from SEQ ID NOs: 162,    165, 166, 167 and 169.-   B1. A method for producing a diacid, comprising:    -   contacting a genetically modified yeast of any one of        embodiments A1 to A77 with a feedstock capable of being        converted by the yeast to a diacid; and    -   culturing the yeast under conditions in which the diacid is        produced from the feedstock.-   B2. The method of embodiment B1, wherein the feedstock comprises one    or more components from a vegetable oil.-   B3. The method of embodiment B1 or B2, wherein the diacid is a C4 to    C24 diacid.-   B4. The method of embodiment B3, wherein the diacid is a C10, C12,    C14, C16, C18 or C20 diacid.-   B5. The method of embodiment B4, wherein the diacid is a C10 diacid.-   B6. The method of embodiment B4, wherein the diacid is a C12 diacid.-   B7. The method of embodiment B4, wherein the diacid is a C18 diacid.-   B8. The method of any one of embodiments B1 to B7, wherein the    diacid contains no unsaturation.-   B9. The method of any one of embodiments B1 to B7, wherein the    diacid contains one or more unsaturations.-   B10. The method of any one of embodiments B1 to B9, wherein the    diacid is the predominant diacid in a mixture of diacids.-   B11. The method of any one of embodiments B1 to B10, wherein the    feedstock comprises a substantially pure oil.-   B12. The method of any one of embodiments B1 to B10, wherein the    feedstock comprises a plurality of fatty acids.-   B13. The method of embodiment B12, wherein the feedstock comprises a    soapstock.-   B14. The method of embodiment B12, wherein the feedstock comprises a    fatty acid distillate.-   B15. The method of any one of embodiments B1 to B14, wherein the    vegetable oil is from a plant chosen from palm, palm kernel,    coconut, soy, safflower, canola or combination thereof.-   C1. A method for producing a diacid by a yeast from a feedstock    toxic to the yeast, comprising:    -   (a) contacting a genetically modified yeast in culture with a        feedstock not substantially toxic to the yeast, thereby        performing an induction; and    -   (b) contacting the yeast after the induction in (a) with a        feedstock toxic to the yeast,    -   whereby a diacid is produced by the yeast from the feedstock        toxic to the yeast in an amount greater than the amount of the        diacid produced from the feedstock toxic to the yeast when the        induction is not performed.-   C2. The method of embodiment C1, wherein the feedstock not    substantially toxic to the yeast has the same number of carbons as    the feedstock toxic to the yeast.-   C3. The method of embodiment C1, wherein the feedstock not    substantially toxic to the yeast has a different number of carbons    compared to the feedstock toxic to the yeast.-   C4. The method of any one of embodiments C1 to C3, wherein the    feedstock not substantially toxic to the yeast comprises a fatty    acid methyl ester.-   C5. The method of any one of embodiments C1 to C3, wherein the    feedstock not substantially toxic to the yeast comprises a free    fatty acid.-   C6. The method of any one of embodiments C1 to C5, wherein the    feedstock not substantially toxic to the yeast comprises more than    twelve carbons.-   D1. An isolated nucleic acid comprising a polynucleotide that    encodes a polypeptide of SEQ ID NO: 148 or 150.-   D2. The isolated nucleic acid of embodiment D1, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 147    or 149.-   D3. An isolated nucleic acid, comprising a polynucleotide that    comprises: the nucleotide sequence of SEQ ID NO: 37 or a nucleotide    sequence having greater than 75% identity to SEQ ID NO: 37.-   D4. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 44; a polypeptide comprising    an amino acid sequence having greater than 71% identity to SEQ ID    NO: 44; or a polypeptide of SEQ ID NO: 44 having 1 to 5 amino acid    substitutions.-   D5. The isolated nucleic acid of embodiment D4, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 43 or    a nucleotide sequence having greater than 69% identity to SEQ ID NO:    43.-   D6. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 46; a polypeptide comprising    an amino acid sequence having greater than 71% identity to SEQ ID    NO: 46; or a polypeptide of SEQ ID NO: 46 having 1 to 5 amino acid    substitutions.-   D7. The isolated nucleic acid of embodiment D6, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 45 or    a nucleotide sequence having greater than 70% identity to SEQ ID NO:    45.-   D8. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 48; a polypeptide comprising    an amino acid sequence having greater than 87% identity to SEQ ID    NO: 48; or a polypeptide of SEQ ID NO: 48 having 1 to 5 amino acid    substitutions.-   D9. The isolated nucleic acid of embodiment D8, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 47 or    a nucleotide sequence having greater than 78% identity to SEQ ID NO:    47.-   D10. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 50; a polypeptide comprising    an amino acid sequence having greater than 80% identity to SEQ ID    NO: 50; or a polypeptide of SEQ ID NO: 50 having 1 to 5 amino acid    substitutions.-   D11. The isolated nucleic acid of embodiment D10, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 49 or    a nucleotide sequence having greater than 75% identity to SEQ ID NO:    49.-   D12. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 67; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 67; or a polypeptide of SEQ ID NO: 67 having 1 to 5 amino acid    substitutions.-   D13. The isolated nucleic acid of embodiment D12, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 68 or    a nucleotide sequence having greater than 97% identity to SEQ ID NO:    68.-   D14. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 74; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 74; or a polypeptide of SEQ ID NO: 74 having 1 to 5 amino acid    substitutions.-   D15. The isolated nucleic acid of embodiment D14, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 73 or    a nucleotide sequence having greater than 97% identity to SEQ ID NO:    73.-   D16. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 76; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 76; or a polypeptide of SEQ ID NO: 76 having 1 to 5 amino acid    substitutions.-   D17. The isolated nucleic acid of embodiment D16, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 75 or    a nucleotide sequence having greater than 99% identity to SEQ ID NO:    75.-   D18. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 3; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 3; or a polypeptide of SEQ ID NO: 3 having 1 to 5 amino acid    substitutions.-   D19. The isolated nucleic acid of embodiment D18, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or    a nucleotide sequence having greater than 99% identity to SEQ ID NO:    2.-   D20. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 5; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 5; or a polypeptide of SEQ ID NO: 5 having 1 to 5 amino acid    substitutions.-   D21. The isolated nucleic acid of embodiment D20, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 4 or    a nucleotide sequence having greater than 98% identity to SEQ ID NO:    4.-   D22. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 7; a polypeptide comprising    an amino acid sequence having greater than 99% identity to SEQ ID    NO: 7; or a polypeptide of SEQ ID NO: 7 having 1 to 5 amino acid    substitutions.-   D23. The isolated nucleic acid of embodiment D22, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 6 or    a nucleotide sequence having greater than 99% identity to SEQ ID NO:    6.-   D24. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 34; a polypeptide comprising    an amino acid sequence having greater than 95% identity to SEQ ID    NO: 34; or a polypeptide of SEQ ID NO: 34 having 1 to 5 amino acid    substitutions.-   D25. The isolated nucleic acid of embodiment D24, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 33 or    a nucleotide sequence having greater than 73% identity to SEQ ID NO:    33.-   D26. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 36; a polypeptide comprising    an amino acid sequence having greater than 94% identity to SEQ ID    NO: 36; or a polypeptide of SEQ ID NO: 36 having 1 to 5 amino acid    substitutions.-   D27. The isolated nucleic acid of embodiment D3, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 35 or    a nucleotide sequence having greater than 73% identity to SEQ ID NO:    35.-   D28. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 129; a polypeptide comprising    an amino acid sequence having greater than 89% identity to SEQ ID    NO: 129; or a polypeptide of SEQ ID NO: 129 having 1 to 5 amino acid    substitutions.-   D29. The isolated nucleic acid of embodiment D28, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 130    or a nucleotide sequence having greater than 84% identity to SEQ ID    NO: 130.-   D30. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 113; a polypeptide comprising    an amino acid sequence having greater than 85% identity to SEQ ID    NO: 113; or a polypeptide of SEQ ID NO: 113 having 1 to 5 amino acid    substitutions.-   D31. The isolated nucleic acid of embodiment D30, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 114    or a nucleotide sequence having greater than 84% identity to SEQ ID    NO: 114.-   D32. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 115; a polypeptide comprising    an amino acid sequence having greater than 97% identity to SEQ ID    NO: 115; or a polypeptide of SEQ ID NO: 115 having 1 to 5 amino acid    substitutions.-   D33. The isolated nucleic acid of embodiment D32, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 116    or a nucleotide sequence having greater than 86% identity to SEQ ID    NO:116-   D34. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 117; a polypeptide comprising    an amino acid sequence having greater than 80% identity to SEQ ID    NO: 117; or a polypeptide of SEQ ID NO: 117 having 1 to 5 amino acid    substitutions.-   D35. The isolated nucleic acid of embodiment D34, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 118    or a nucleotide sequence having greater than 80% identity to SEQ ID    NO: 118.-   D36. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 119; a polypeptide comprising    an amino acid sequence having greater than 84% identity to SEQ ID    NO: 119; or a polypeptide of SEQ ID NO: 119 having 1 to 5 amino acid    substitutions.-   D37. The isolated nucleic acid of embodiment D36, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 120    or a nucleotide sequence having greater than 76% identity to SEQ ID    NO: 120.-   D38. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 121; a polypeptide comprising    an amino acid sequence having greater than 81% identity to SEQ ID    NO: 121; or a polypeptide of SEQ ID NO: 121 having 1 to 5 amino acid    substitutions.-   D39. The isolated nucleic acid of embodiment D38, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 122    or a nucleotide sequence having greater than 74% identity to SEQ ID    NO: 122.-   D40. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 123; a polypeptide comprising    an amino acid sequence having greater than 90% identity to SEQ ID    NO: 123; or a polypeptide of SEQ ID NO: 123 having 1 to 5 amino acid    substitutions.-   D41. The isolated nucleic acid of embodiment D40, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 124    or a nucleotide sequence having greater than 82% identity to SEQ ID    NO: 124.-   D42. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 125; a polypeptide comprising    an amino acid sequence having greater than 80% identity to SEQ ID    NO: 125; or a polypeptide of SEQ ID NO: 125 having 1 to 5 amino acid    substitutions.-   D43. The isolated nucleic acid of embodiment D42, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 126    or a nucleotide sequence having greater than 77% identity to SEQ ID    NO: 126.-   D44. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 127; a polypeptide comprising    an amino acid sequence having greater than 81% identity to SEQ ID    NO: 127; or a polypeptide of SEQ ID NO: 127 having 1 to 5 amino acid    substitutions.-   D45. The isolated nucleic acid of embodiment D44, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 128    or a nucleotide sequence having greater than 78% identity to SEQ ID    NO: 128.-   D46. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 96; a polypeptide comprising    an amino acid sequence having greater than 85% identity to SEQ ID    NO: 96; or a polypeptide of SEQ ID NO: 96 having 1 to 5 amino acid    substitutions.-   D47. The isolated nucleic acid of embodiment D46, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 95 or    a nucleotide sequence having greater than 72% identity to SEQ ID    NO:95-   D48. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 139; a polypeptide comprising    an amino acid sequence having greater than 76% identity to SEQ ID    NO: 139; or a polypeptide of SEQ ID NO: 139 having 1 to 5 amino acid    substitutions.-   D49. The isolated nucleic acid of embodiment D48, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 140    or a nucleotide sequence having greater than 77% identity to SEQ ID    NO: 140.-   D50. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 141; a polypeptide comprising    an amino acid sequence having greater than 83% identity to SEQ ID    NO: 141; or a polypeptide of SEQ ID NO: 141 having 1 to 5 amino acid    substitutions.-   D51. The isolated nucleic acid of embodiment D50, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 142    or a nucleotide sequence having greater than 73% identity to SEQ ID    NO: 142.-   D52. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 90; a polypeptide comprising    an amino acid sequence having greater than 95% identity to SEQ ID    NO: 90; or a polypeptide of SEQ ID NO:90 having 1 to 5 amino acid    substitutions.-   D53. The isolated nucleic acid of embodiment D52, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 89 or    a nucleotide sequence having greater than 81% identity to SEQ ID NO:    89.-   D54. An isolated nucleic acid comprising a polynucleotide that    encodes: the polypeptide of SEQ ID NO: 40; a polypeptide comprising    an amino acid sequence having greater than 92% identity to SEQ ID    NO: 40; or a polypeptide of SEQ ID NO: 40 having 1 to 5 amino acid    substitutions.-   D55. The isolated nucleic acid of embodiment D54, wherein the    polynucleotide comprises the nucleotide sequence of SEQ ID NO: 39 or    a nucleotide sequence having greater than 84% identity to SEQ ID NO:    39.-   D56. An isolated nucleic acid, comprising a polynucleotide that    comprises: the nucleotide sequence of SEQ ID NO: 166 or a nucleotide    sequence having greater than 84% identity to SEQ ID NO: 166.-   D57. An isolated nucleic acid, comprising a polynucleotide that    comprises: the nucleotide sequence of SEQ ID NO: 167 or a nucleotide    sequence having greater than 85% identity to SEQ ID NO: 167.-   D58. An isolated nucleic acid, comprising a polynucleotide that    comprises: the nucleotide sequence of SEQ ID NO: 164 or a nucleotide    sequence having greater than 92% identity to SEQ ID NO: 169.-   D59. The isolated nucleic acid of any one of embodiments D3 to D58,    wherein at least one of the 1 to 5 amino acid substitutions is    conservative.-   D60. The isolated nucleic acid of any one of embodiments D3 to D58,    wherein at least one of the 1 to 5 amino acid substitutions is    non-conservative.-   E1. The isolated nucleic acid of any one of embodiments D1 to D60,    which is an expression vector.-   E2. A cell comprising a nucleic acid of any one of embodiments D1 to    E1.-   E3. The cell of embodiment E2, which is a bacterium.-   E4. The cell of embodiment E2, which is a yeast.-   E5. The cell of embodiment E4, which is a Candida spp. yeast.-   E6. The cell of embodiment E5, wherein the Candida spp. yeast is    chosen from C. tropicalis and C. viswanathii.-   E7. The cell of embodiment E6, wherein the Candida spp. yeast is a    genetically modified ATCC20336 yeast.-   E8. The cell of embodiment E4, which is chosen from a Yarrowia spp.    yeast, Pichia spp. yeast, Saccharomyces spp. yeast and Kluyveromyces    spp. yeast.-   E9. The cell of embodiment E8, which is chosen from Y.    lipolytica, P. pastoris, P. membranifaciens, P. kluyveri, P.    guilliermondii, P. heedii, P. subpelliculosa, S. cerevisiae, S.    bayanus, S. pastorianus, S. carlsbergensis, K. lactis and K.    marxianus.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

1-168. (canceled)
 169. A method for producing a diacid, comprising:contacting a genetically modified yeast with a feedstock capable ofbeing converted by the yeast to a diacid; and culturing the yeast underconditions in which the diacid is produced from the feedstock, whereinthe genetically modified yeast comprises: one or more geneticmodifications that substantially block beta oxidation activity; and oneor more genetic modifications that increase ADH2a alcohol dehydrogenaseactivity, wherein ADH2a alcohol dehydrogenase comprises the amino acidsequence of SEQ ID NO:
 115. 170. The method of claim 169, wherein thegenetically modified yeast comprises one or more genetic modificationsthat increase a monooxygenase activity.
 171. The method of claim 170,wherein the monooxygenase comprises CYP52A12, CYP52A13, CYP52A14,CYP52A15, CYP52A16, CYP52A17, CYP52A18, CYP52A19 or CYP52A20.
 172. Themethod of claim 169, wherein the genetically modified yeast comprisesone or more genetic modifications that increase a monooxygenasereductase activity.
 173. The method of claim 172, wherein themonooxygenase reductase is cytochrome p450 reductase B (CPRB).
 174. Themethod of claim 169, wherein the genetically modified yeast comprisesone or more genetic modifications that disrupt activity of a POX4polypeptide or a POX5 polypeptide.
 175. The method of claim 169, whereinthe genetically modified yeast is a Candida spp. yeast.
 176. The methodof claim 175, wherein the Candida spp. yeast is chosen from C.tropicalis and C. viswanathii.
 177. The method of claim 175, wherein theCandida spp. yeast is a genetically modified ATCC20336 yeast.
 178. Themethod of claim 169, wherein the feedstock comprises a substantiallypure oil.
 179. The method of claim 169, wherein the feedstock comprisesa plurality of fatty acids.
 180. The method of claim 169, wherein thefeedstock comprises one or more components from a vegetable oil. 181.The method of claim 180, wherein the vegetable oil is from a plantchosen from palm, palm kernel, coconut, soy, safflower, canola orcombination thereof.
 182. The method of claim 169, wherein the diacidcontains one or more unsaturations.
 183. The method of claim 169,wherein the diacid is a C8, C10, C12, C14, C16, C18 or C20 diacid. 184.The method of claim 183, wherein the diacid is a C12 diacid.
 185. Themethod of claim 169, wherein the diacid is dodecanedioic acid or12-hydroxydodecanoic acid.